Anti-lap antibody variants and uses thereof

ABSTRACT

Provided herein are anti-LAP antibodies (e.g., recombinant humanized, chimeric, and human anti-LAP antibodies) or antigen binding fragments thereof which have therapeutically beneficial properties, such as binding specifically to LAP-TGFβ1 on cells but not to LAP-TGFβ1 in extracellular matrix, as well as compositions including the same. Also provided are uses of these antibodies or antigen binding fragments in therapeutic applications, such as in the treatment of cancer, and diagnostic applications.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/744,045, filed Oct. 10, 2018, U.S. Provisional PatentApplication No. 62/750,065, filed Oct. 24, 2018, and U.S. ProvisionalPatent Application No. 62/757,519, filed Nov. 8, 2018, each of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-LAP antibodies or antigen bindingfragments thereof. Another aspect of the invention relates tocompositions and kits comprising the anti-LAP antibodies or antigenbinding fragments. Another aspect of the invention relates to methodsfor treating diseases, for example cancer, by administering theantibodies or antigen binding fragments.

BACKGROUND

Transforming growth factor beta 1 (TGFβ1) is synthesized as apro-protein complex, in which the mature cytokine is caged within LAP(latency associated peptide), which is the latency associated peptide ofTGFβ1. The LAP-TGFβ1 complex is disulfide bonded to one of fivecurrently known anchor proteins: Glycoprotein A repetitions predominant(GARP), Leucine-rich repeat-containing protein 33 (LRRC33),Latent-transforming growth factor beta-binding protein 1 (LTBP1),Latent-transforming growth factor beta-binding protein 3 (LTBP3), andLatent-transforming growth factor beta-binding protein 4 (LTBP4). Theseanchor proteins localize latent TGFβ1 in particular sites and onparticular cells within the body.

GARP, also referred to as leucine-rich repeat protein 32 or LRRC32, is atransmembrane protein that anchors LAP-TGFβ1 to the surface oflymphocytes, most notably regulatory T cells. GARP is also expressed onplatelets, B cells, NK cells, fibroblasts, mesenchymal stromal cells,mesenchymal stem cells, and endothelial cells and also governs LAP-TGFβ1expression on those cell types. LRRC33 is a transmembrane protein thatis reported to anchor LAP-TGFβ1 to the surface of myeloid cells, mostnotably macrophages, dendritic cells, and myeloid derived suppressorcells (MDSCs). LTBP1, LTBP3, and LTBP4 are secreted molecules thatanchor LAP-TGFβ1 into the extracellular matrix (ECM).

Although LAP binding agents have been used in the art as tools toidentify certain cell populations, little is known about LAP's relevancein disease states.

The location of the LAP-TGFβ1 complex is of critical biological andclinical importance because, once the mature TGFβ1 cytokine, which has ashort half-life in solution, is released, it acts locally, either in anautocrine or near paracrine fashion. Therefore, the anchor proteins area principal mechanism whereby latent TGFβ1 is staged in a specificlocation, awaiting the release of the potent mature cytokine to act onthe local tissue.

LAP-TGFβ1 has different functions when expressed in different locations.For example, LAP-TGFβ1 anchored by LTBPs in the extracellular matrix isof primary importance for tissue homeostasis. In this regard, Xu et al.(Bone Research 2018; 6:2) noted that “the TGF-β complex is more like amolecular sensor that responds instantly to ECM perturbations throughthe release of an active ligand that exerts physiological effects at acellular level, thus ensuring normal tissue homeostasis.”

Alterations in LAP-TGFβ1 incorporation into the extracellular matrix areknown to result in human disease. For example, deletion of LTBP-3 inboth mice and humans results in similar defects in both bone and dentalformation. LTBP-3 defects are also associated with the aortic dilationseen in Marfan syndrome (Rifkin et al., Matrix Biol 2018; 71-72:90-99).These effects are believed to be due to aberrant direct effects of TGFβ1in the local extracellular matrix (Xu et al, Bone Research 2018; 6:2).

In contrast to anchor proteins that localize LAP-TGFβ1 to theextracellular matrix, LAP-TGFβ1 anchored by GARP is of primaryimportance for the immunosuppressive function of regulatory T cells(Edwards et al, Eur J Immunol 2016; 46:1480-9) and of suppressive B cellsubpopulations (Wallace et al, JCI Insight 2018; 3:e99863). Some tumorshave also been shown to express GARP, allowing them to locally expressTGFβ and directly suppress the immune system in the tumormicroenvironment and support their own growth (Metelli et al, Journal ofHematology & Oncology 2018; 11:24).

LAP-TGFβ1 anchored to myeloid cells is of primary importance for theimmunosuppressive function of MDSCs (Zhang H et al, Frontiers inImmunology 2017; 8:1-15) and of M2 macrophages (Zhang et al., Oncotarget2017; 8:99801-15). According to a recent study, myeloid cells have beenshown to use the anchor protein LRRC33 to anchor latent TGFβ to the cellsurface (Qin et al, Cell 2018; 174:1-16).

Recent developments in cancer therapy have focused on harnessing apatient's immune system by, e.g., activation of exhausted immune cellpopulations, vaccination, and removal of immunosuppressive cellpopulations. Given the ongoing need for improved strategies fortargeting (and diagnosing) diseases such as cancer, novel agents andmethods that are useful for these purposes are desired.

SUMMARY

An aspect of the invention provided herein is a construct (e.g.,polynucleotide, expression vector and host cell), protein or peptidecomprising any of the sequences described herein, for example, the aminoacid sequences found in tables such as Table 34. Provided herein areantibodies and antigen binding fragments thereof that bind LAPcomprising the structural and functional features specified below (e.g.,any one of the amino acid sequences of SEQ ID NOs: 16-197, 214, 216-240,242-245, 248, 249 and 255 in Table 34). For example, the antibodies andantigen binding fragments comprise the amino acid sequences described inthe tables herein, e.g., SEQ ID NOs: 16-197, 214, and 216-255. Invarious embodiments, the LAP comprises a complex and/or an epitopecomprising LAP and a TGFβ (e.g., TGFβ1). In various embodiments, theepitope is described in examples herein, e.g., Examples 19-23.

An aspect of the invention provides isolated monoclonal antibodies(e.g., recombinant humanized, chimeric, and human antibodies) whichexhibit therapeutically advantageous patterns of binding to LAP-TGFβ1(e.g., human LAP-TGFβ1) and functional properties compared to prioranti-LAP antibodies. In one embodiment, the anti-LAP antibodiesselectively bind to LAP-TGFβ1 on cells (e.g., immune cells and otherimmunosuppressive cells) but not to LAP-TGFβ1 in the extracellularmatrix, and thus are able to target a broad range of clinically relevantcell types while sparing the natural function/activation of LAP-TGFβ1 inthe extracellular matrix. Because TGFβ acts in an autocrine ornear-paracrine manner, selective binding to specific cell populationswill result in inhibition of the production of mature TGFβ in theimmediate proximity of the indicated cell population. Accordingly, theantibodies described herein provide the clinical benefit of inhibitingTGFβ activation and release of the mature cytokine in a highlyselective, cell-specific manner. In some embodiments, the anti-LAPantibodies are of an isotype with active effector function and enhancedbinding of a specific anti-LAP antibody to a given cell population willresult in increased depletion of that cell population by ADCC or CDC.Accordingly, anti-LAP antibodies disclosed herein are ideal for treatinga broad variety of diseases, including cancers and other diseasesinvolving immunosuppressive cells, both in monotherapy and combinationwith other immunomodulatory or therapeutic agents (e.g., immunecheckpoint inhibitors).

In another aspect of the invention, provided herein is an antibody(e.g., recombinant humanized, chimeric, domain, or human antibody) orantigen binding fragment thereof which specifically binds to LAPcomprising:

(a) a heavy chain variable region comprising complementarity determiningregion (CDR) 1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 16, 26, and 18, respectively, and a light chainvariable region comprising CDR1, CDR2, and CDR3 regions comprising theamino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively;

(b) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(c) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(d) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and68, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(e) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and68, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(f) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively; or

(g) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively.

In various embodiments, the antibody is a humanized antibody, chimericantibody, or human antibody.

In various embodiments, the administering step (e.g., in a method oftreating or diagnosing a subject) is performed with the antibody. Invarious embodiments, the antibody is a humanized antibody, chimericantibody or human antibody. In various embodiments, the LAP is humanLAP, cynomolgus monkey (cyno) LAP, rat LAP, and/or mouse LAP. In variousembodiments, the administering step (e.g., in a method of treating ordiagnosing a subject) is performed with the antigen binding fragment.

In various embodiments, the constant region of the antibody is a humanIgG1 constant region. For example, the IgG1 constant region comprisesthe amino acid sequence set forth in a table disclosed herein, (e.g.,Table 34). For example, the IgG1 constant region comprises the aminoacid sequence set forth in SEQ ID NOs: 196, 244, or 245. In variousembodiments, the constant region of the antibody is a human IgG4constant region. For example, the human IgG4 constant region comprisesthe amino acid sequence set forth in SEQ ID NO: 197.

In another aspect of the invention, provided herein is an isolatedantibody or antigen binding fragment which specifically binds to humanLAP and comprises heavy and light chain variable region sequences whichare at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to theamino acid sequences selected from the group consisting of: (a) SEQ IDNOs: 42 and 52, respectively; (b) SEQ ID NOs: 101 and 104, respectively;(c) SEQ ID NOs: 98 and 104, respectively; (d) SEQ ID NOs: 133 and 154,respectively; and (e) SEQ ID NOs: 218 and 154, respectively.

In another aspect of the invention, provided herein is an isolatedantibody or antigen binding fragment which specifically binds to humanLAP and comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 218 or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 218 with 1, 2, or 3amino acid substitutions; and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 154 or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 154 with 1, 2,or 3 amino acid substitutions. In various embodiments, at least onesubstitution is located within a CDR. In various embodiments, at leastone substitution is located within a framework region. In variousembodiments, at least one substitution is located within at least oneCDR and at least one the framework region. In various embodiments, anyand/or all of the at least one substitution is located and/or foundwithin the framework region(s). In various embodiments, the antibody isa humanized antibody, chimeric antibody, or human antibody. Anotheraspect of the invention provided herein is an isolated antibody orantigen binding fragment which specifically binds to human LAP andcomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 218 or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 218 with 1-5, 5-10, 10-15, 15-20,or 20-25 amino acid substitutions; and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 154 or a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 154with 1-5, 5-10, 10-15, 15-20, or 20-25 amino acid substitutions. Forexample, at least one substitution is located in at least one CDRregion. In various embodiments, the at least one substitution is locatedwithin multiple CDRs. In various embodiments, the at least onesubstitution is located within at least one framework region. In variousembodiments, the at least one substitution is located within at leastone CDR and in at least one framework region. In various embodiments,any and/or all of the at least one substitution is located and/or foundwithin the framework region(s). In various embodiments, the antibody isa humanized antibody, chimeric antibody, or human antibody.

Another aspect of the invention provided herein is an isolated antibodyor antigen binding fragment which specifically binds to human LAP,wherein the antibody or antigen binding fragment comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 218,wherein the antibody or antigen binding fragment comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 154. Invarious embodiments, the antibody is a humanized antibody, chimericantibody, or human antibody.

Another aspect of the invention provided herein is an isolated antibodyor antigen binding fragment which specifically binds to human LAP,wherein the antibody or antigen binding fragment comprises a heavy chainvariable region consisting of the amino acid sequence of SEQ ID NO: 218,wherein the antibody or antigen binding fragment comprises a light chainvariable region consisting of the amino acid sequence of SEQ ID NO: 154.In various embodiments, the antibody is a humanized antibody, chimericantibody, or human antibody.

Another aspect of the invention provided herein is an isolated antibodyor antigen binding fragment which specifically binds to human LAP,wherein the antibody or antigen binding fragment comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 219, wherein theantibody or antigen binding fragment comprises a light chain comprisingthe amino acid sequence of SEQ ID NO: 155. In various embodiments, theantibody is a humanized antibody, chimeric antibody, or human antibody.

Another aspect of the invention provided herein is an isolated antibodyor antigen binding fragment which specifically binds to human LAP,wherein the antibody or antigen binding fragment comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 219, wherein theantibody or antigen binding fragment comprises a light chain consistingof the amino acid sequence of SEQ ID NO: 155. In various embodiments,the antibody is a humanized antibody, chimeric antibody, or humanantibody. In another aspect of the invention, provided herein is anisolated antibody or antigen binding fragment which specifically bindsto human LAP and comprises heavy and light chain sequences which are atleast 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acidsequences selected from the group consisting of: (a) SEQ ID NOs: 43 and53, respectively; (b) SEQ ID NOs: 45 and 53, respectively; (c) SEQ IDNOs: 102 and 105, respectively; (d) SEQ ID NOs: 103 and 105,respectively; (e) SEQ ID NOs: 99 and 105, respectively; (f) SEQ ID NOs:100 and 105, respectively; (g) SEQ ID NOs: 134 and 155, respectively;(h) SEQ ID NOs: 135 and 155, respectively; (i) SEQ ID NOs: 219 and 155,respectively; and (j) SEQ ID NOs: 220 and 155, respectively. Forexample, at least one substitution is located in at least one CDRregion. In various embodiments, the at least one substitution is locatedwithin multiple CDRs. In various embodiments, the at least onesubstitution is located within at least one framework region. In variousembodiments, the at least one substitution is located within at leastone CDR and in at least one framework region. In various embodiments,any and/or all of the at least one substitution is located and/or foundwithin the framework region(s). In various embodiments, the antibody isa humanized antibody, chimeric antibody, or human antibody.

In another aspect of the invention, provided herein is an isolatedantibody or antigen binding fragment which binds to human LAP andcomprises a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively, wherein the antibody furthercomprises a human IgG1 constant region. In various embodiments, theantibody is a humanized antibody, chimeric antibody, or human antibody.

In another aspect of the invention, provided herein is an isolatedantibody or antigen binding fragment which binds to human LAP andcomprises a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising amino acid sequences that are at least 80%, 85%, 90%,95%, 98%, 99%, or 100% identical to the amino acid sequences SEQ ID NOs:110, 120, and 112, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising amino acid sequencesthat are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to theamino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively,wherein the antibody further comprises a human IgG1 constant region. Invarious embodiments, the antibody is a humanized antibody, chimericantibody, or human antibody.

In another aspect of the invention, provided herein is an isolatedantibody or antigen binding fragment which binds to human LAP andcomprises a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively, wherein the antibody furthercomprises a mutant human IgG4 constant region comprising the amino acidsequence of SEQ ID NO: 197. In various embodiments, the antibody is ahumanized antibody, chimeric antibody, or human antibody.

An aspect of the invention provides an anti-LAP antibody orantigen-binding fragment thereof described herein (e.g., 20E6 andhumanized versions thereof described in Table 34) is in association withan isolated antibody comprising an immunoglobulin heavy chain comprisingthe amino acid sequence of SEQ ID NO: 240 and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO: 241.

An aspect of the invention provides anti-LAP antibody or antigen-bindingfragment thereof described herein (e.g., 20E6 and humanized versionsthereof described in Table 34) is in association with an isolatedantibody comprising an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 246 and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 247.

In another aspect of the invention provided herein is an isolatedantibody or antigen binding fragment which binds to the same epitope onLAP as the anti-LAP antibodies or antigen binding fragments describedherein. In another aspect provided herein is an isolated antibody orantigen binding fragment which binds to the same amino acids or groupsof amino acids on LAP as the anti-LAP antibodies or antigen bindingfragments described herein. For example, the epitope (e.g., LAP and aLAP complex comprising LAP and TBFβ1), antibody, or antigen bindingfragment has the characteristics described herein, such as Tables 25,26, 27, 28, 29, and/or 30. In some embodiments, the anti-LAP antibodybinds to specific amino acids of human LAP, for example amino acids31-40, 274-280, and 340-343 of human LAP-TGFβ1 (SEQ ID NO: 1), e.g., asassessed by at least one structural analytical method such ascrystallography and/or cryo-EM. In some embodiments, the anti-LAPantigen binding fragment binds to specific amino acids of human LAP, forexample amino acids 31-40, 274-280, and 340-343 of human LAP-TGFβ1 (SEQID NO: 1), e.g., as assessed by at least one structural analyticalmethod such as crystallography and/or cryo-EM. In various embodiments,the antibody or the antigen binding fragment binds to one or more aminoacids within the recited amino acids, i.e., one or more amino acidswithin amino acids 31-40, 274-280, and 340-343 of human LAP-TGFβ1 (SEQID NO: 1). In some embodiments, the isolated antibody or antigen bindingfragment binds to one or more residues of residues 31-40, 274-280, and340-343 of human LAP-TGFβ1 (SEQ ID NO: 1), or binds to one or moreresidues of residues 31-43, 272-283, and 340-344 of human LAP-TGFβ1 (SEQID NO: 1). In some embodiments, the anti-LAP antibody or antigen bindingfragment thereof binds to a specific region or regions of LAP-TGFβ1, forexample, Region 1, Region 2, Region 3, and/or Region 4 as shown in FIG.34, e.g., as assessed by at least one structural analytical method suchas HDX-MS.

In some embodiments, the antibody or antigen binding fragment binds tohuman LAP (e.g., with a K_(D) of about 11 nm, with a K_(D) of 11 nM orless, or with a K_(D) of 10 nM or less). In various embodiments, theantibody or antigen binding fragment binds to LAP (e.g., human, cyno,rat or mouse) with a K_(D) of 60 nM or less, 50 nM or less, 40 nM orless, 30 nM or less, 20 nM or less, or 10 nM or less. In someembodiments, the antibody or antigen binding fragment binds to human LAP(e.g., with a K_(D) of less than 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or10 nM). In some embodiments, the antibody or antigen binding fragmentbinds to human LAP with a K_(D) of about 40-60 nM, or about 50-60 nM. Invarious embodiments, the K_(D) is determined by Octet binding analysis.In various embodiments, the K_(D) is determined by BIACORE® surfaceplasmon resonance (referred to interchangeably as “BiaCore” and“BIACore”) binding analysis. In various embodiments, the antibody orantigen binding fragment has a binding affinity described herein, e.g.,Tables 31-32. In some embodiments, the antibody or antigen bindingfragment inhibits TGFβ1 activation. In various embodiments, the antibodyor antigen binding fragment inhibits integrin activation of TGFβ and/orrelease of human LAP from the LAP-TGFβ1 complex. In some embodiments,the antibody or antigen binding fragment binds to both human and murineLAP. In some embodiments, the antibody or antigen binding fragment bindsto human LAP in the absence of an anchor protein. In some embodiments,the antibody or antigen binding fragment binds or is determined to bindto LAP-TGFβ1 complexed with an anchor protein (e.g., GARP, LRRC33) onimmunosuppressive cells, but does not bind to the anchor protein or toan epitope composed of residues of both LAP-TGFβ and the anchor protein.Immunosuppressive cells include, for example, suppressive T cells (e.g.,regulatory T cells, activated T cells), cancer-associated fibroblasts,M2 macrophages, cancer cells expressing LAP-TGFβ1, and/or monocyticmyeloid-derived suppressor cells. In some embodiments, the antibody orantigen binding fragment does not bind free TGFβ1 or empty LAP. In someembodiments, the antibody or antigen binding fragment does not bind toLAP in extracellular matrix. In some embodiments, the antibody orantigen binding fragment does not bind or is determined to not to bindto LAP complexed with LTBP1, LTBP3 and/or LTBP4. In some embodiments,the antibody or antigen binding fragment binds or is determined to bindto human LAP-TGFβ1 comprising K27C and Y75C mutations and/or all or aportion of (e.g., within) residues 82-130 of human LAP-TGFβ1 (SEQ ID NO:1), but not human LAP-TGFβ1 comprising the Y74T mutation.

In some embodiments, the antibody or antigen binding fragment binds oris determined to bind to both GARP-positive immunosuppressive cells andGARP-negative immunosuppressive cells. In some embodiments, the antibodyor antigen binding fragment binds or is determined to bind to platelets,but does not cause platelet aggregation or platelet degranulation.

In some embodiments, the antibody is an IgG1, IgG2, IgG3, IgG4, IgD,IgA, IgE, or IgM antibody, or variant thereof. In some embodiments, theantibody is a chimeric, domain, humanized, or human antibody.

In any of the above-mentioned embodiments, the antibody or antigenbinding fragment thereof can comprise any of the variable light chainsdescribed herein and light chain constant domain described herein (e.g.,a human light chain constant domain). For example, the light chainconstant domain is recited in Table 34. In one embodiment, the antibodyor antigen binding fragment thereof comprises a human kappa light chainconstant domain or a variant thereof. In various embodiments, thevariant comprises up to 1-25 modified amino acid substitutions (e.g., 20substitutions). In another embodiment, the antibody or antigen bindingfragment thereof comprises a human lambda light chain constant domain ora variant thereof. In various embodiments, the variant comprises up to1-25 modified amino acid substitutions (e.g., substitutions). In oneembodiment, the antibody or antigen binding fragment thereof comprises ahuman kappa light chain constant domain comprising the amino acidsequence of SEQ ID NO: 256.

In another aspect of the invention, provided herein is a bispecificantibody comprising a first binding region with a specificity for LAP ofan anti-LAP antibody described herein, and a second binding region ortherapeutic agent which binds to another antigen, e.g., atumor-associated antigen, CD4, CD8, CD45, CD56, CD14, CD16, CD19, CD11b,CD25, CD20, CD22, CD30, CD38, CD114, CD23, CD73, CD163, CD206, CD203,CD200R or CD39. In various embodiments, the second binding region ortherapeutic agent which binds to a receptor protein.

In another aspect of the invention, provided herein is animmunoconjugate comprising an anti-LAP antibody or antigen bindingfragment described herein linked to a detectable moiety, a bindingmoiety, a labeling moiety, and/or a biologically active moiety, e.g., abispecific molecule and/or a bifunctional molecule. For example, thebiologically active moiety comprises a receptor trap construct.

In another aspect of the invention, provided herein is a nucleic acid(one or more nucleic acids) comprising a nucleotide sequence thatencodes the heavy and/or light chain variable region of an anti-LAPantibody or antigen binding fragment described herein, as well asexpression vector(s) comprising the same, and cells transformed with theexpression vector(s). In another aspect, provided herein is a nucleicacid (one or more nucleic acids) comprising a nucleotide sequence thatencodes the heavy chain and/or light chain of an anti-LAP antibody orantigen binding fragment described herein, as well as expressionvector(s) comprising the same, and cells transformed with the expressionvector(s).

In another aspect of the invention, provided herein is a pharmaceuticalcomposition comprising an anti-LAP antibody or antigen binding fragmentdescribed herein and a pharmaceutically acceptable carrier. In someembodiments, the composition comprises one or more additionaltherapeutic agents, such as an anti-cancer agent, a chemotherapeuticagent, an immunomodulatory agent (e.g., an immunostimulatory agent orimmunosuppressive agent), an anti-inflammatory agent, and/or an immunecheckpoint blocker (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody,an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody,and an anti-TIM3 antibody). For example, the PD-1 antibody ispembrolizumab.

In an embodiment, an anti-LAP antibody or antigen-binding fragmentthereof described herein (e.g., 20E6 and humanized versions thereofdescribed in Table 34) is in association with an isolated antibodycomprising an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 240 and an immunoglobulin light chain comprisingthe amino acid sequence of SEQ ID NO: 241. SEQ ID NOs: 240 and 241correspond to the heavy chain and light chain sequences ofpembrolizumab.

In an embodiment, an anti-LAP antibody or antigen-binding fragmentthereof described herein (e.g., 20E6 and humanized versions thereofdescribed in Table 34) is in association with an isolated antibodycomprising an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 246 and an immunoglobulin light chain comprisingthe amino acid sequence of SEQ ID NO: 247. SEQ ID NOs: 246 and 247correspond to the heavy and light chain sequences of pembrolizumab.

In another aspect of the invention, provided herein are kits comprisingan anti-LAP antibody or antigen binding fragment described herein andinstructions for use.

In another aspect of the invention, provided herein is a method ofmaking an antibody that specifically binds to LAP comprising: (a)immunizing an animal with a polypeptide comprising an epitope on humanLAP recognized by 28G11, (b) selecting from the immunized animal anantibody that binds to the same epitope as 28G11, and (c) isolating theantibody selected from step (b). In some embodiments, the antibody bindsto a human LAP complex comprising TGFβ1. In some embodiments, theantibody binds to all or a portion (e.g., within) residues 82-130 ofhuman LAP.

In another aspect of the invention, provided herein is a method ofselectively inhibiting TGFβ1 activation on cells (e.g.,immunosuppressive cells such as suppressive T cells (e.g., regulatory Tcells, activated T cells), M2 macrophages, cancer cells expressingLAP-TGFβ1, cancer-associated fibroblasts, mesenchymal stromal cellsmesenchymal stem cells, and/or monocytic myeloid-derived suppressorcells), but not TGFβ1 activation on extracellular matrix, comprisingadministering to the subject any anti-LAP antibody or antigen bindingfragment, bispecific molecule, immunoconjugate, and/or pharmaceuticalcomposition described herein.

In another aspect of the invention, provided herein is a method oftreating cancer comprising administering to a subject in need thereof atherapeutically effective amount of any anti-LAP antibody or antigenbinding fragment, bispecific molecule, immunoconjugate, and/orpharmaceutical composition described herein.

In some embodiments, the cancer is characterized by abnormal TGFβactivity. In some embodiments, the cancer is associated withinfiltration of cluster of differentiation 4 (CD4)+ regulatory T cells,cluster of differentiation 8 (CD8)+ regulatory T cells, regulatory Bcells, myeloid-derived suppressor cells, tumor-associated macrophages,cancer-associated fibroblasts, and/or innate lymphoid cells.

In some embodiments, the cancer is breast cancer, bladder cancer,uterine/cervical cancer, ovarian cancer, prostate cancer, testicularcancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer,colorectal cancer, colon cancer, kidney cancer, head and neck cancer,lung cancer, stomach cancer, germ cell cancer, bone cancer, livercancer, thyroid cancer, skin cancer, neoplasm of the central nervoussystem, lymphoma, leukemia, myeloma, sarcoma, or myelodysplasticsyndromes.

In some embodiments of the methods described above, one or moreadditional therapies is administered, for example, radiation therapy,chemotherapy, an immune checkpoint inhibitor (e.g., an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4antibody, an anti-TIGIT antibody, and an anti-TIM3 antibody),immunostimulatory therapy, immunosuppressive therapy, cell therapy, anda therapeutic agent (e.g., anti-cancer agent, a chemotherapeutic agent,an immunosuppressive agent, an immunomodulatory agent, and ananti-inflammatory agent).

In another aspect of the invention, provided herein is a method ofdetecting LAP comprising contacting a sample (e.g., a biological sample)with any anti-LAP antibody or antigen binding fragment, bispecificmolecule, immunoconjugate, and/or pharmaceutical composition describedherein, and detecting the complex.

In another aspect of the invention, provided herein is a method ofdiagnosing a cancer associated with regulatory T cell infiltrationcomprising contacting a biological sample from a patient afflicted withthe cancer with any anti-LAP antibody or antigen binding fragment,bispecific molecule, immunoconjugate, and/or pharmaceutical compositiondescribed herein, wherein positive staining with the antibody or antigenbinding fragment, bispecific molecule, immunoconjugate, and/orpharmaceutical composition indicates the cancer is associated withregulatory T cell infiltration.

In another aspect of the invention, provided herein is a method ofdiagnosing a cancer associated with GARP-negative suppressive cellscomprising contacting a biological sample from a patient afflicted withthe cancer with any anti-LAP antibody or antigen binding fragment,bispecific molecule, immunoconjugate, and/or pharmaceutical compositiondescribed herein which binds to GARP-negative suppressive cells, whereinpositive staining with the antibody or antigen binding fragment,bispecific molecule, immunoconjugate, and/or pharmaceutical compositionand negative staining with an anti-GARP antibody indicates the cancer isassociated with GARP-negative suppressive cells.

In another aspect of the invention, provided herein is a method ofselecting a patient afflicted with cancer for treatment with an anti-LAPantibody or antigen binding fragment, bispecific molecule,immunoconjugate, and/or pharmaceutical composition described hereincomprising contacting a biological sample from the patient with theantibody or antigen binding fragment, bispecific molecule,immunoconjugate, and/or pharmaceutical composition, wherein positivestaining with the antibody or antigen binding fragment indicates thecancer is amenable to treatment with the antibody.

In another aspect of the invention, provided herein is a method ofdetermining the response of a patient afflicted with cancer to treatmentwith an anti-LAP antibody or antigen binding fragment described hereincomprising contacting a biological sample from the patient with theantibody or antigen binding fragment, wherein reduced staining with theantibody or antigen binding fragment, bispecific molecule,immunoconjugate, and/or pharmaceutical composition indicates the canceris responding to treatment with the antibody.

In another aspect of the invention, provided herein is a method ofmaking an antibody that specifically binds to the same epitope on humanLAP recognized by 28G11 comprising immunizing an animal with animmunogen comprising a peptide, wherein the peptide comprises theepitope recognized by 28G11, selecting from the immunized animal anantibody that binds to the same epitope as 28G11, and obtaining anantibody that binds to the same epitope as 28G11.

In various embodiments, the human LAP comprises a complex comprisinghuman LAP and TGFβ1.

Another aspect of the invention are uses of any of the anti-LAPantibodies or antigen binding fragments, bispecific molecules,immunoconjugates, and/or pharmaceutical compositions described hereinfor selectively inhibiting TGFβ1 activation on immunosuppressive cells,but not TGFβ1 activation on extracellular matrix; treating cancer;diagnosing a cancer (e.g., a cancer associated with regulatory T cellinfiltration or GARP-negative suppressive cells); selecting a patientafflicted with cancer; and determining the response of a patientafflicted with cancer to treatment with the anti-LAP antibodiesdescribed herein. Also provided are uses of any of the anti-LAPantibodies or antigen binding fragments, bispecific molecules,immunoconjugates, and/or pharmaceutical compositions described hereinfor preparing a medicament to selectively inhibit TGFβ1 activation onimmunosuppressive cells, but not TGFβ1 activation on extracellularmatrix, and to treat cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F are graphs showing the binding of antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, 24E3_hIgG1, and 2C9_(hyb) onnon-transfected HT1080 cells (HT 1080-null) and HT1080 cellsoverexpressing human LAP-TGFβ1 (HT1080-huB1), human LAP-TGFβ2(HT1080-huB2), human LAP-TGFβ3 (HT1080-huB3), and murine LAP-TGFβ1(HT1080-muB1). [TGFβ2=transforming growth factor beta-2;TGFβ3=transforming growth factor beta-3]

FIGS. 2A-2F are graphs showing the binding of antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, 24E3_hIgG1, and 2C9_(hyb) to theindicated LAP-TGFβ variants. Black bars correspond to the indicatedantibody binding to HT1080 cells over-expressing human LAP-TGFβ1. Graybars correspond to negative controls samples where no anti-LAP antibodywas added. HT1080-huB1: HT1080 cells overexpressing LAP-TGFβ1,HT1080-K27C_Y75C: HT1080 cells overexpressing LAP-TGFβ1 with K27C andY75C mutations (mutations that prevent TGFβ1 activation by integrins;“closed” conformation”), HT1080-Y74T: HT1080 cells overexpressingLAP-TGFβ1 with a Y74T mutation (mutation that favors spontaneous releaseof TGFβ1 (“open conformation”); HT1080-ch2.3: HT1080 cellsoverexpressing chimeric LAP-TGFβ1 in which exon 2.3 (residues 131-164)of human LAP-TGFβ1 have been replaced with corresponding residues fromchicken LAP-TGFβ1 (UniProt accession #H9CX01); HT1080-ch4: HT1080 cellsoverexpressing chimeric LAP-TGFβ1 in which exon 4 (residues 183-208) ofhuman LAP-TGFβ1 has been replaced with exon 4 from chicken LAP-TGFβ1.

FIGS. 3A-3F are graphs showing the binding of antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, 24E3_hIgG1, and 2C9_(hyb) to theindicated LAP-TGFβ variants. Black bars correspond to the indicatedantibody binding to HT1080 cells over-expressing human LAP-TGFβ1. Graybars correspond to negative controls samples where no anti-LAP antibodywas added. HT1080-huB1: HT1080 cells overexpressing LAP-TGFβ1,HT1080-emptyLAP: HT1080 cells overexpressing LAP which does not includethe mature TGFβ1 cytokine, HT1080-ch2.2: HT1080 cells overexpressingchimeric LAP-TGFβ1 in which exon 2.2 (residues 108-130) of humanLAP-TGFβ1 has been replaced with exon 2.2 from chicken LAP-TGFβ1.

FIG. 4 is a graph showing the binding of antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, 24E3_hIgG1, and 1D11 to mature TGFβ(i.e., TGFβ without LAP), as measured by an ELISA assay in whichinhibition of signal reflects binding to mature TGFβ.

FIGS. 5A and 5B are sets of graphs showing the effects of 28G11 on TGFβ1activation in P3U1 cells expressing human LAP-TGFβ1 (FIG. 5A) and murineLAP-TGFβ1 (FIG. 5B).

FIGS. 6A-6F are graphs showing the effects of antibodies) 17G8 (FIG. 6Afor human LAP-TGFβ1 and FIG. 6B for mouse LAP-TGFβ1), 24E3 (FIG. 6C forhuman LAP-TGFβ1 and FIG. 6D for murine LAP-TGFβ1), 22F9 (FIG. 6E forhuman LAP-TGFβ1), 20E6 (FIG. 6F for human LAP-TGFβ1 on TGFβ1 activationin P3U1 cells expressing human or mouse LAP-TGFβ1. FIGS. 6G-6L aregraphs showing the effects of 28G11_IgG2a (FIG. 6G), 20E6_IgG2a (FIG.6H), 22F9_IgG2a (FIG. 6I), 24E3_hIgG1 (FIG. 6J), 17G8_hIgG1 (FIG. 6K),and 20E6_H0.2aL1_hIgG1 (FIG. 6L) on TGFβ1 activation in P3U1 cellsexpressing human or mouse LAP-TGFβ1.

FIG. 7 is a graph showing binding of the indicated anti-LAP antibodiesto extracellular matrix (ECM) deposited by P3E11 cells. Three cell typeswere tested: P3E11 cells without LAP-TGFβ, P3E11 cells expressing humanLAP-TGFβ1, and P3E11 cells expressing murine LAP-TGFβ1. Antibodies wereused at a concentration of 2 μg/mL.

FIG. 8 is a graph showing the dose-response relationship in binding ofthe indicated anti-LAP antibodies (i.e., antibodies 28G11, 22F9, 20E6,17G8, and 24E3) to human platelets.

FIGS. 9A-9E are graphs showing the effects of anti-LAP antibodies28G11_hyb (FIG. 9A), 20E6_IgG2a (FIG. 9B), 22F9_IgG2a (FIG. 9C),17G8_hIgG1 (FIG. 9D), and 24E3_hIgG1 (FIG. 9E) on human plateletdegranulation.

FIGS. 10A and 10B are graphs showing the binding of the indicatedanti-LAP antibodies to THP-1 cells. THP cell binding is expressed as thepercentage (%) of LAP+ cells in FIG. 10A and as median fluorescentintensity (MFI) in FIG. 10B. FIG. 10C is a graph showing the binding ofthe indicated anti-LAP antibodies to THP-1 cells. THP cell binding isexpressed as the % of LAP+θcells. FIG. 10D shows dot plots for thebinding of the indicated anti-LAP antibodies to THP-1 cells. FIGS. 10Eand 10F are graphs showing the binding of the indicated anti-LAPantibodies to U937 cells. U937 cell binding is expressed as the % ofLAP+ cells in FIG. 10E and as MFI in FIG. 10F. FIG. 10G shows dot plotsfor the binding of the indicated anti-LAP antibodies to U937 cells.

FIG. 11 is a graph showing the binding of anti-LAP antibodies 28G11,22F9, and 20E6 to regulatory T cells, CD8 cells, M2 macrophages, andM-MDSCs from CT26 TILs, expressed as the percentage of positive cells.

FIG. 12 is a graph showing the ability of humanized 28G11 variants tocompete with murine 28G11 parental antibody for binding to P3U1 cellsover-expressing human GARP and LAP-TGFβ1. H0-H2 are variant humanizedheavy chains, and L0-L3 are variant humanized light chains.

FIG. 13 is a graph showing the binding of the indicated humanized 28G11variants to P3U1 cells over-expressing human GARP and LAP-TGFβ1.

FIGS. 14A-14D are graphs showing the inhibition of TGFβ1 activation bythe indicated humanized 28G11 variants by ELISA.

FIG. 15A is a graph showing the effects that various combinations ofreversions in the humanized 28G11_H2L3 variant back to human residueshave on binding to HT1080 cells overexpressing human LAP-TGFβ1. Thevarious antibodies tested are listed as HC variant_LC variant. FIG. 15Bis a graph showing the effects that the 28G11_H2.1L3 and 28G11_H2aL3avariants have on binding to HT1080 cells overexpressing human LAP-TGFβ1.Black bars correspond to antibody binding to HT1080 cellsover-expressing human LAP-TGFβ1. Gray bars correspond to antibodybinding to control HT1080 cells which do not over-express humanLAP-TGFβ1.

FIG. 16 is a graph showing the binding of the indicated humanized 22F9variants to HT1080 cells over-expressing human LAP-TGFβ1. Black barscorrespond to antibody binding to HT1080 cells over-expressing humanLAP-TGFβ1. Gray bars correspond to antibody binding to control HT1080cells which do not over-express human LAP-TGFβ1.

FIGS. 17A and 17B are chromatograms from size exclusion high-performanceliquid chromatography (SE-HPLC) for the indicated antibody 22F9 andvariants.

FIG. 18A is a graph showing the effects of reverting murine residues inthe heavy chain of 22F9_H5L0 back to corresponding human residues onbinding to HT1080 cells over-expressing human LAP-TGFβ1. FIG. 18B is agraph showing the effects of substitutions to remove potentialdeamidation and/or isomerization sites in 22F9_H5L0 on binding to HT1080cells over-expressing human LAP-TGFβ1. 22F9_H5.2L0 corresponds to22F9_H5L0 with the double N54Q/D102A mutation. FIG. 18C is a graphshowing the binding of 22F9_H0.1, 22F9_H1.1, 22F9_H2.1, and 22F9_H3.1variants to HT1080 cells over-expressing human LAP-TGFβ1. Black barscorrespond to antibody binding to HT1080 cells over-expressing humanLAP-TGFβ1. Gray bars correspond to antibody binding to control HT1080cells which do not over-express human LAP-TGFβ1.

FIGS. 19A and 19B are graphs showing the binding of the indicatedhumanized 20E6 variants to human LAP-TGFβ1. Black bars correspond toantibody binding to HT1080 cells over-expressing human LAP-TGFβ1. Graybars correspond to antibody binding to control HT1080 cells which do notover-express human LAP-TGFβ1.

FIG. 20A is a graph showing the effects of murine back-substitutions tothe L1 light chain of 20E6 on binding to human LAP-TGFβ1. FIG. 20B is agraph showing the binding of the 20E6 H0 heavy chain variant withreduced immunogenicity on binding to human LAP-TGFβ1. Black barscorrespond to antibody binding to HT1080 cells over-expressing humanLAP-TGFβ1. Gray bars correspond to antibody binding to control HT1080cells which do not over-express human LAP-TGFβ1.

FIGS. 21A-21C are bio-layer interferometry curves showing the binding of20E6_H0.2aL1_hIgG1 antibody to Fc fusions comprising either humanLAP-TGFβ1, LAP-TGFβ2, or LAP-TGFβ3.

FIGS. 22A-22D are graphs showing the binding of multiple heavy and lightchain CDR variants of 20E6_H0.2aL1_hIgG1 antibody to human LAP-TGFβ1.

FIGS. 23A and 23B are graphs showing the binding of F(ab′)2 fragmentsand Fab′ fragments of 20E6_H0.2aL1_hIgG1 to P3U1 cells overexpressingGARP and LAP-TGFβ1.

FIG. 24 is a series of bio-layer interferometry curves showing thebinding of MHG8 (a GARP-specific murine IgG2a) antibody, 16F4 (ananti-LAP) antibody, and humanized 20E6 antibody to soluble LAP-TGFβ1,sGARP-LAP-TGFβ1, and ECR3E-LAP-TGFβ1 complexes.

FIG. 25 is a schematic of the competition experiment used to comparebinding epitopes of murine 28G11, 16F4, and MHG8, as described inExample 17.

FIG. 26A shows an example of a cryo-EM map used to build the humanized20E6-Fab/LAP-TGFβ1 model. FIG. 26B is a plot of the FSC curve versusresolution. FIG. 26C is the B-factor estimation using a Guinier plot.

FIG. 27A is a cryo-EM map of the humanized 20E6 antibody complexed withLAP-TGFβ1 at 3.1 Å resolution. FIG. 27B shows the electron density forPDB entry 5jxe at 2.9 A resolution.

FIG. 28A shows interactions between LAP and the VL and VH of thehumanized 20E6 Fab. FIG. 28B shows interactions between TGFβ1 and thehumanized 20E6 VL and VH portions of the Fab. FIG. 28C and FIG. 28D showadditional interactions between LAP-TGFβ1 and the 20E6 VL portion of the20E6 Fab. Constructs and interactions are shown in grayscale.

FIG. 29A shows interactions between LAP and the VL and VH of the 28G11humanized Fab. FIG. 29B shows interactions between TGFβ1 and thehumanized 28G11 VL and VH portions of the Fab. FIG. 29C shows therepositioning of CDRs in humanized 28G11 and humanized 20E6. FIG. 29Dshows that Q56 in humanized 28G11 is ideally positions to interact withthe TGFβ1 loop spanning residues V342-K344. FIG. 29E shows that, inhumanized 28G11, a Tyr residue is replaced by Gly, resulting in loss ofinteractions with the side chain of V341. Constructs and interactionsare shown in grayscale.

FIG. 30A shows the 2Mab:2TGFβ1 complex identified in cryo-EM (Mab refersto 22F9 Fab). Constructs are: LAP-TGFβ1 dimer; 22F9-Fabs; 22F9-Fcs. FIG.30B provides examples of the 2D classes. Constructs and interactions areshown in grayscale.

FIG. 31A shows interactions between LAP and the VL and VH portions ofthe 22F9 Fab. FIG. 31B shows interactions between TGFβ and 22F9 VL andVH. FIG. 31C shows an overlay of 22F9-Fab+LAP-TGFβ1 complex andhumanized 20E6-Fab+LAP-TGFβ1 (Fabs) and the different orientation ofhumanized 20E6-Fabs and 22F9-Fabs with respect to the antigen. FIG. 31Dand FIG. 31E show additional interactions between LAP-TGFβ1 and portionsof the 20E6 Fab. Constructs and interactions are shown in grayscale.

FIG. 32 is a H/D Difference Plot showing deuterium uptake protectionupon humanized 20E6 antibody binding to human LAP-TGFβ1 protein.

FIG. 33 is a H/D Difference Plot showing deuterium uptake protectionupon antibody binding to human LAP-TGFβ1 protein.

FIG. 34 is a sequence alignment between human LAP-TGFβ1 and mouseLAP-TGFβ1. Epitopes identified are all in regions that are homologousbetween human and mouse LAP-TGFβ1. The human LAP-TGFβ1 sequence in thefigure corresponds to SEQ ID NO: 257. The mouse LAP-TGFβ1 sequence inthe figure corresponds to SEQ ID NO: 7.

FIGS. 35A and 35B are size-exclusion chromatograms for human LAP-TGFβ1,GARP-LAP-TGFβ1, humanized 20E6, and complexes. The elution time for eachsample is consistent with the gel filtration standard. Molecular weightreported was determined from the light scattering detector.

FIGS. 36A-36F are a series of graphs that show the effects of anti-LAPantibodies 28G11 and 16B4 in combination with an anti-PD-1 antibody ontumor volume in a syngeneic CT26 colorectal cancer tumor model. The datashown in the figures are: FIG. 36A (anti-PD-1 antibody alone), FIG. 36B(28G11_IgG2a+anti-PD-1 antibody), FIG. 36C (IgG2a isotype control), FIG.36D (anti-PD-1 antibody alone), FIG. 36E (16B4_IgG2a alone), and FIG.36F (16B4_IgG2a+anti-PD-1 antibody). The anti-PD-1 antibody was a ratanti-PD-1 (clone RMP1-14)-IgG2a antibody.

FIG. 37 is a graph that shows the effects of anti-LAP antibody 28G11 incombination with an anti-PD-1 antibody on tumor volume in a syngeneicEMT6 breast cancer tumor model. The anti-PD-1 antibody was a ratanti-PD-1 (clone RMP1-14)-IgG2a antibody. The statistical test used wastwo-way ANOVA.

FIG. 38 is a graph that shows the effects of anti-LAP antibody 22F9 incombination with an anti-PD-1 antibody on tumor volume in a syngeneicEMT6 breast cancer tumor model. The anti-PD-1 antibody was a ratanti-PD-1 (clone RMP1-14)-IgG2a antibody. The statistical test used wastwo-way ANOVA.

FIG. 39 is a graph that shows the effects of anti-LAP antibody 20E6 incombination with an anti-PD-1 antibody on tumor volume in a syngeneicEMT6 breast cancer tumor model. The anti-PD-1 antibody was a ratanti-PD-1 (clone RMP1-14)-IgG2a antibody. The statistical test used wastwo-way ANOVA.

FIG. 40 is a graph that shows the effects of anti-LAP antibodies28G11_IgG2a and 16B4_IgG2a, and the anti-TGFβ antibody 1D11_IgG1, asmonotherapy on lung nodule counts in the 4T1 breast cancer tumormetastasis model (p<0.05, unpaired T test following removal ofoutliers).

FIGS. 41A and 41B are graphs that show the effects of anti-LAP antibody28G11_IgG2a in combination with 12 Gy (FIG. 41A) and 20 Gy (FIG. 41B)radiation on tumor volume in the syngeneic CT26 tumor model. Thestatistical test used was 2-way ANOVA. ****P<0001, ***P=0.0004.

FIGS. 42A-42C are graphs that show the effects of anti-LAP antibody28G11_IgG2a on CD73 expression in M-MDSCs (FIG. 42A), M2 macrophages(FIG. 42B), and dendritic cells (FIG. 42C), with or without 12 Gy or 20Gy radiation.

FIGS. 43A-43H are a series of graphs that show effects of anti-LAPantibodies 28G11-mIgG2a and 20E6-mIgG2a alone and in combination withanti-PD-1 antibody on tumor volume in a syngeneic EMT6 mouse breastcancer tumor model. The data shown in the figures are: FIG. 43 A is agraph summary of efficacy of 28G11-mIgG2a and FIG. 43B is a graphsummary of efficacy of 20E6-mIgG2a. Also shown is a tumor volume datafor control antibodies (FIG. 43C), antibody 20E6-mIgG2a (FIG. 43D),antibody 28G11-mIgG2a (FIG. 43E), anti-PD-1 antibody (FIG. 43F), acombination of antibody 20E6-mIgG2a and anti-PD-1 antibody (FIG. 43G),and antibody 28G11-mIgG2a and anti-PD-1 antibody (FIG. 43H).

FIGS. 44A-44D are a series of Biacore graphs showing binding of anti-LAPF(ab′) to human LAP-TGFβ isoforms 1, 2, and 3.

FIGS. 45A-45D are a series of Biacore graphs showing binding of anti-LAPF(ab′) to human, cynomolgus monkey, rat, and mouse LAP-TGFβ1.

FIG. 46 is a graph showing inhibition of integrin (avb6) activation ofLAP-TGFβ1 using 20E6_mIgG2a antibody, isotype control antibody oranti-αVβ6 (10D5) antibody.

DETAILED DESCRIPTION Definitions

In order for the following detailed description to be readilyunderstood, certain terms are first defined. Additional definitions areprovided throughout.

“Abnormal” in the context of the activity, level or expression of amolecule means that the activity, level or expression is outside of thenormal activity, level or expression for that molecule. “Normal” in thecontext of activity, level or expression refers to the range ofactivity, level or expression of the protein found in a population ofhealthy, gender- and age-matched subjects. The minimal size of thishealthy population may be determined using standard statisticalmeasures, e.g., the practitioner could take into account the incidenceof the disease in the general population and the level of statisticalcertainty desired in the results. Preferably, the normal range foractivity, level or expression of a biomarker is determined from apopulation of subjects (e.g., at least five, ten or twenty subjects),more preferably from a population of at least forty or eighty subjects,and even more preferably from more than 100 subjects.

As used herein, “Latency associated peptide” or “LAP” refers to theamino-terminal domain of the human TGFβ1 precursor peptide and has theamino acid sequence set forth in SEQ ID NO: 2. “LAP-TGFβ1” and“LAP/TGFβ1” are used interchangeably herein to refer to the human TGFβ1precursor peptide (which includes the TGFβ1 cytokine) and includes theamino acid sequence of SEQ ID NO: 1 (Uniprot sp|P01137TGFB1_HUMAN withsignal sequence removed).

LAP can also refer to the amino-terminal domains of the human TGFβ2precursor peptide (LAP region: SEQ ID NO: 4, LAP-TGFβ2: SEQ ID NO: 3)and human TGFβ3 precursor peptide (LAP region: SEQ ID NO: 6, LAP-TGFβ2:SEQ ID NO: 5), as well as their counterparts from other species (e.g.,mouse TGFβ1 precursor peptide (mouse LAP region: SEQ ID NO: 8; mouseLAP-TGFβ1: SEQ ID NO: 7), mouse TGFβ2 precursor peptide (mouse LAPregion: SEQ ID NO: 10; mouse LAP-TGFβ2: SEQ ID NO: 9), and mouse TGFβ3precursor peptide (mouse LAP region: SEQ ID NO: 12; mouse LAP-TGFβ3: SEQID NO: 11)) and other naturally occurring allelic, splice variants, andprocessed forms thereof. LAP is synthesized as a complex with TGFβ. LAPin the absence of mature TGFβ is referred to as “empty LAP.” Unlessotherwise specified, “empty LAP” as used herein refers to LAPoriginating from the N-terminal domain of human TGFβ1. In addition toresidues on LAP, the anti-LAP antibodies described herein may also bindto residues of mature TGFβ within the LAP-TGFβ1 complex.Notwithstanding, in all cases, the antibody at least binds to residuesin the LAP portion of the complex.

As used herein “free TGFβ1” refers to the mature TGFβ1 cytokine, i.e.,TGFβ1 that is not complexed with LAP.

As used herein, “anchor protein” refers to a protein that anchorsLAP-TGFβ to a cell surface or to the extracellular matrix. Exemplaryanchor proteins include GARP, LRRC33, LTBP1, LTBP3, and LTBP4. GARP andLRRC33 are proteins that anchor LAP-TGFβ to the surface of cells, andLTBP1, LTBP3, and LTBP4 are proteins that anchor LAP-TGFβ to theextracellular matrix.

The term “antibody” as used herein includes whole antibodies and anyantigen binding fragments (i.e., “antigen-binding portions”) or singlechains thereof. An “antibody” refers, in one embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Incertain naturally occurring antibodies, the heavy chain constant regionis comprised of three domains, CH1, CH2, and CH3. In certain naturallyoccurring antibodies, each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁴ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less,even more preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸M and 10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens.

The phrase “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human and/or mouse LAP). Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments of an antibody include (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the V_(H) and CH1 domains; (iv) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbfragment (Ward et al., (1989) Nature 341:544-546), which consists of aV_(H) domain; and (vi) an isolated complementarity determining region(CDR) or (vii) a combination of two or more isolated CDRs which mayoptionally be joined by a synthetic linker. Furthermore, although thetwo domains of the Fv fragment, V_(L) and V_(H), are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

Antibody fragments within the scope of the present invention alsoinclude F(ab′)2 fragments which may be produced by enzymatic cleavage ofan IgG by, for example, pepsin. Fab fragments may be produced by, forexample, reduction of F(ab′)2 with dithiothreitol or mercaptoethylamine.A Fab fragment is a VL-CL chain appended to a VH-CH1 chain by adisulfide bridge. A F(ab′)2 fragment is two Fab fragments which, inturn, are appended by two disulfide bridges. The Fab portion of anF(ab′)2 molecule includes a portion of the Fc region between whichdisulfide bridges are located.

The term “acceptor human framework” refers to a framework comprising theamino acid sequence of a light chain variable domain (V_(L)) frameworkor a heavy chain variable domain (V_(H)) framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may have the same amino acid sequence as thenaturally-occurring human immunoglobulin framework or human consensusframework, or it may have amino acid sequence changes compared towild-type naturally-occurring human immunoglobulin framework or humanconsensus framework. In some embodiments, the number of amino acidchanges are 10, 9, 8, 7, 6, 5, 4, 3, or 2, or 1. In some embodiments,the V_(L) acceptor human framework is identical in sequence to the V_(L)human immunoglobulin framework sequence or human consensus frameworksequence.

A “multispecific antibody” is an antibody (e.g., bispecific antibodies,tri-specific antibodies) that recognizes two or more different antigensor epitopes.

The term “binding protein” as used herein also refers to a non-naturallyoccurring (or recombinant) protein that specifically binds to at leastone target antigen.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Bifunctionalantibodies include, for example, heterodimeric antibody conjugates(e.g., two antibodies or antibody fragments joined together with eachhaving different specificities), antibody/cell surface-binding moleculeconjugates (e.g., an antibody conjugated to a non-antibody molecule suchas a receptor), and hybrid antibodies (e.g., an antibody having bindingsites for two different antigens).

The term “recombinant antibody,” refers to antibodies that are prepared,expressed, created or isolated by recombinant means, such as (a)antibodies isolated from an animal (e.g., a mouse) that is transgenic ortranschromosomal for immunoglobulin genes (e.g., human immunoglobulingenes) or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialantibody library (e.g., containing human antibody sequences) using phagedisplay, and (d) antibodies prepared, expressed, created or isolated byany other means that involve splicing of immunoglobulin gene sequences(e.g., human immunoglobulin genes) to other DNA sequences. Suchrecombinant antibodies may have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies can be subjected to in vitromutagenesis and thus the amino acid sequences of the V_(H) and V_(L)regions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline V_(H) and V_(L) sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

A “human” antibody refers to an antibody having variable regions inwhich both the framework and CDR regions are derived from human germlineimmunoglobulin sequences. Furthermore, if the antibody contains aconstant region, the constant region also is derived from human germlineimmunoglobulin sequences. Also encompassed are antibodies derived fromhuman germline immunoglobulin sequences that include normal somatichypermutations which alter the germline immunoglobulin sequencesrelative to the wild-type germline immunoglobulin sequences.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Anyadditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one or more species and the constant regionsare derived from another species, such as an antibody in which thevariable regions are derived from a mouse antibody and the constantregions are derived from a human antibody. See U.S. Pat. No. 4,816,567;and Morrison et al., (1984) Proc. Natl. Acad Sci. USA 81: 6851-6855.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies in the population are substantiallysimilar and bind the same epitope(s) (e.g., the antibodies display asingle binding specificity and affinity), except for possible variantsthat may arise during production of the monoclonal antibody, suchvariants generally being present in minor amounts.

“Monoclonal” indicates the character of the antibody as having beenobtained from a substantially homogenous population of antibodies, anddoes not require production of the antibody by any particular method.

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a particular epitope. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., (1975) Nature 256: 495, or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., (1991) Nature 352: 624-628 andMarks et al., (1991) J. Mol. Biol. 222: 581-597.

Antigen binding fragments (including scFvs) of such immunoglobulins arealso encompassed by the term “monoclonal antibody” as used herein.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations, which typically include differentantibodies directed against different epitopes on the antigen, eachmonoclonal antibody is directed against a single epitope. Monoclonalantibodies can be prepared using any art recognized technique and thosedescribed herein such as, for example, a hybridoma method, a transgenicanimal, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), orusing phage antibody libraries using the techniques described in, forexample, U.S. Pat. No. 7,388,088 and PCT Pub. No.

WO 00/31246). Monoclonal antibodies include chimeric antibodies, humanantibodies, and humanized antibodies and may occur naturally or beproduced recombinantly.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

As used herein, the term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linker.For a review of sFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONALANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, NewYork, pp. 269-315.

The monoclonal antibodies herein also include camelized single domainantibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci.26:230; Reichmann et al. (1999)J. Immunol. Methods 231:25; WO 94/04678;WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two V_(H) domains withmodifications such that single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described more fully in, e.g., EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants generally seeHolliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta (2006) Adv Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes, suchas substitutions, deletions and insertions, glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc may beutilized to alter the half-life of antibodies in therapeutic antibodies,and a longer half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

The term “fully human antibody” refers to an antibody that compriseshuman immunoglobulin protein sequences only. A fully human antibody maycontain murine carbohydrate chains if produced in a mouse, in a mousecell, or in a hybridoma derived from a mouse cell. Similarly, “mouseantibody” refers to an antibody which comprises mouse immunoglobulinsequences only.

As used herein, the term “hypervariable region” (sometimes referred toas the “variable region”) refers to the amino acid residues of anantibody that are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or “CDR” (e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97(CDRL3) in the light chain variable domain and residues 31-35 (CDRH1),50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain;Kabat et al., (1991) Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md.) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk, (1987) J. Mol. Biol. 196: 901-917).

As used herein, the term “framework” or “FR” residues refers to thosevariable domain residues other than the hypervariable region residuesdefined herein as CDR residues. The residue numbering above relates tothe Kabat numbering system and does not necessarily correspond in detailto the sequence numbering in the accompanying Sequence Listing. Aminoacid residues in antibodies can also be defined using other numberingsystems, such as Chothia, enhanced Chothia, IMGT, Kabat/Chothiacomposite, Honegger (AHo), Contact, or any other conventional antibodynumbering scheme.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

As used herein, “isotype” refers to the antibody class (e.g., IgG(including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 andIgA2), IgD, and IgE antibody) that is encoded by the heavy chainconstant region genes of the antibody.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include Clq binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated phagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc region,” “Fc domain,” or “Fc” refers to the C-terminal region ofthe heavy chain of an antibody. Thus, an Fc region comprises theconstant region of an antibody excluding the first constant regionimmunoglobulin domain (e.g., CH1 or CL).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., human LAP-TGFβ1) to which an immunoglobulin or antibodyspecifically binds. Epitopes can be formed both from contiguous aminoacids (usually a linear epitope) or noncontiguous amino acids juxtaposedby tertiary folding of the protein (usually a conformational epitope).Epitopes formed from contiguous amino acids are typically, but notalways, retained on exposure to denaturing solvents, whereas epitopesformed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 aminoacids in a unique spatial conformation.

The term “epitope mapping” refers to the process of identifying themolecular determinants on the antigen involved in antibody-antigenrecognition. Methods for determining what epitopes are bound by a givenantibody are well known in the art and include, for example,immunoblotting and immunoprecipitation assays, wherein overlapping orcontiguous peptides from, e.g., LAP-TGFβ1 are tested for reactivity witha given antibody (e.g., anti-LAP antibody); x-ray crystallography;antigen mutational analysis, two-dimensional nuclear magnetic resonance;yeast display; and hydrogen/deuterium exchange-mass spectrometry(HDX-MS) (see, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66, G. E. Morris, Ed. (1996)). See also Champe et al.(1995) J. Biol. Chem. 270:1388-1394.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment or samesegments of amino acid residues, as determined by a given method.Techniques for determining whether antibodies bind to the “same epitopeon LAP-TGFβ1” with the antibodies described herein include, for example,epitope mapping methods, such as x-ray analyses of crystals ofantigen:antibody complexes, which provides atomic resolution of theepitope, and HDX-MS. Other methods monitor the binding of the antibodyto antigen fragments (e.g. proteolytic fragments) or to mutatedvariations of the antigen where loss of binding due to a modification ofan amino acid residue within the antigen sequence is often considered anindication of an epitope component, such as alanine scanning mutagenesis(Cunningham & Wells (1985) Science 244:1081), yeast display of mutanttarget sequence variants, or analysis of chimeras (e.g., as described inExamples 2 and 3). In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest to affinity isolate specific short peptidesfrom combinatorial phage display peptide libraries. Antibodies havingthe same V_(H) and V_(L) or the same CDR1, 2 and 3 sequences areexpected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known binding competition experiments, e.g., BIACORE®surface plasmon resonance (SPR) analysis. In certain embodiments, anantibody competes with, and inhibits binding of another antibody to atarget by at least 50%, 60%, 70%, 80%, 90% or 100%. The level ofinhibition or competition may be different depending on which antibodyis the “blocking antibody” (i.e., the antibody that when combined withan antigen blocks another immunologic reaction with the antigen).Competition assays can be conducted as described, for example, in EdHarlow and David Lane, Cold Spring Harb. Protoc. 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlowand David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., USA 1999. Competing antibodies bind to the same epitope, anoverlapping epitope, or to adjacent epitopes (e.g., as evidenced bysteric hindrance). Two antibodies “cross-compete” if antibodies blockeach other both ways by at least 50%, i.e., regardless of whether one orthe other antibody is contacted first with the antigen in thecompetition experiment.

Competitive binding assays for determining whether two antibodiescompete or cross-compete for binding include competition for binding tocells expressing LAP-TGFβ1, e.g., by flow cytometry. Other methodsinclude: surface plasmon resonance (SPR) (e.g., BIACORE®), solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli et al., Methods in Enzymology 9:242 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986));solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press (1988)); solid phase direct label RIA using 1-125 label(see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directlabeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody (i)binds with an equilibrium dissociation constant (K_(D)) of approximatelyless than 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower when determined by, e.g., surface plasmonresonance (SPR) using a predetermined antigen as the analyte and theantibody as the ligand, or Scatchard analysis of binding of the antibodyto antigen positive cells, and (ii) binds to the predetermined antigenwith an affinity that is at least two-fold greater than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. Any K_(D) greaterthan about 10⁻⁴ M is generally considered to indicate nonspecificbinding.

The term “k_(assoc)” or “k_(a)”, as used herein, refers to theassociation rate of a particular antibody-antigen interaction, whereasthe term “k_(dis)” or “k_(d),” as used herein, is intended to refer tothe dissociation rate of a particular antibody-antigen interaction. Theterm “K_(D)”, as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of k_(d) to k_(a) (i.e.,k_(d)/k_(a)) and is expressed as a molar concentration (M). K_(D) valuesfor antibodies can be determined using methods well established in theart. A preferred method for determining the K_(D) of an antibody is byusing surface plasmon resonance, preferably using a biosensor systemsuch as a Biacore® system or flow cytometry and Scatchard analysis, orbio-layer interferometry.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody refers to the concentration of an antibody that induces aresponse that is 50% of the maximal response, i.e., halfway between themaximal response and the baseline.

The term “cross-reacts,” as used herein, refers to the ability of anantibody described herein to bind to LAP-TGFβ1 from a different species.For example, an antibody described herein that binds human LAP-TGFβ1 mayalso bind another species of LAP-TGFβ1 (e.g., murine LAP-TGFβ1, ratLAP-TGFβ1, or cynomolgus monkey LAP-TGFβ1). Cross-reactivity may bemeasured by detecting a specific reactivity with purified antigen inbinding assays (e.g., SPR, ELISA, bio-layer interferometry) or bindingto, or otherwise functionally interacting with, cells physiologicallyexpressing LAP-TGFβ1 (e.g., HT1080 cells overexpressing LAP-TGFβ1).Methods for determining cross-reactivity include standard binding assaysas described herein, for example, by bio-layer interferometry or flowcytometric techniques.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody fragments (e.g., V_(H),V_(L), CDR3), is intended to refer to a nucleic acid molecule in whichthe nucleotide sequences are essentially free of other genomicnucleotide sequences, e.g., those encoding antibodies that bind antigensother than LAP, which other sequences may naturally flank the nucleicacid in human genomic DNA.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

Also provided are “conservative sequence modifications” of the sequencesset forth herein, i.e., amino acid sequence modifications which do notabrogate the binding of the antibody encoded by the nucleotide sequenceor containing the amino acid sequence, to the antigen. Such conservativesequence modifications include conservative nucleotide and amino acidsubstitutions, as well as, nucleotide and amino acid additions anddeletions. For example, modifications can be introduced into a sequencein a table herein (e.g., Table 34) by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an anti-LAP antibody ispreferably replaced with another amino acid residue from the same sidechain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad Sci. USA 94:412-417 (1997)). Alternatively, inanother embodiment, mutations can be introduced randomly along all orpart of an anti-LAP antibody coding sequence, such as by saturationmutagenesis, and the resulting modified anti-LAP antibodies can bescreened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 80% to 85%, 85% to 90% or 90% to 95%, and more preferably at leastabout 98% to 99.5% of the nucleotides. Alternatively, substantialhomology exists when the segments will hybridize under selectivehybridization conditions, to the complement of the strand. Forpolypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 80% to 85%, 85% to 90%, 90% to 95%, and more preferably at leastabout 98% to 99.5% of the amino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=#ofidentical positions/total #of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or two amino acid sequences canalso be determined using the algorithm of E. Meyers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and may be a cell into whicha recombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “inhibition” as used herein, refers to any statisticallysignificant decrease in biological activity, including partial and fullblocking of the activity. For example, “inhibition” can refer to astatistically significant decrease of about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in biologicalactivity.

As used herein, “TGFβ1 activation” refers to the release of the maturecytokine TGFβ1 from the latent complex made up of LAP and TGFβ1. Thereare many mechanisms known to induce TGFβ1 activation (see Robertson I B,Rifkin D B. Unchaining the beast; insights from structural andevolutionary studies on TGFβ1 secretion, sequestration, and activation.Cytokine Growth Factor Rev. 2013 August; 24(4):355-72). The maturecytokine can be detected using a specific ELISA or similar detectionmethodology or through the use of a reporter cell line that expresses aTGFβ receptor.

For example, as used herein, the term “inhibits TGFβ1 activation”includes any measurable decrease in TGFβ1 activation, e.g., aninhibition of TGFβ1 activation by at least about 10%, for example, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 99%, or about 100%, relative to acontrol (e.g., a control antibody). The inhibition may be specific to asingle mechanism of TGFβ1 activation or may be generalizable to allmechanisms of TGFβ1 activation. As used herein, the term “inhibits TGFβ1activation” includes inhibition of at least one activation mechanism.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject with a tumor or cancer ora subject who is predisposed to having such a disease or disorder, ananti-LAP antibody (e.g., anti-human LAP antibody) described herein, inorder to prevent, cure, delay, reduce the severity of, or ameliorate oneor more symptoms of the disease or disorder or recurring disease ordisorder, or in order to prolong the survival of a subject beyond thatexpected in the absence of such treatment.

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

As used herein, “immune cell” refers to the subset of blood cells knownas white blood cells, which include mononuclear cells such aslymphocytes, monocytes, macrophages, and granulocytes.

As used herein, “immunosuppressive cell” refers to a cell thatcontributes to or promotes an immunosuppressive tumor microenvironment.The presence of a population of immunosuppressive cells in a tumormicroenvironment increases the tumor's resistance to an immune response,resulting in tumor protection, tumor escape, and/or tumor metastasis.Unless countered in some manner, these immunosuppressive cells candecrease the efficacy of immune-mediated anti-cancer treatments.Exemplary immunosuppressive cells include cancer-associated fibroblasts,myeloid-derived suppressor cells, regulatory T cells (Tregs), tumorcells expressing LAP, and immunosuppressive macrophages. These celltypes can be identified by one skilled in the art using, e.g., flowcytometry to identify markers of Tregs (e.g., CD4, FoxP3, CD127, andCD25), macrophages (e.g., CSF-IR, CD203, CD206, CD163, IL-10, and TGFβ),cancer associated fibroblasts (e.g., alpha smooth muscle actin,fibroblast activation protein, tenascin-C, periostin, NG2, vimentin,desmin, PDGFR alpha and beta, FSP-1, ASPN, and STC1), andmyeloid-derived suppressor cells (e.g., CD11b, CD33, CD14, or CD15, andlow levels of HLA DR). It is understood that immunosuppressive cells mayalso be important in suppressing the immune system in other diseasestates.

As used herein, “suppressive T cells” refer to T cells that contributeto or promote an immunosuppressive microenvironment. Exemplarysuppressive T cells include CD4+ regulatory T cells and CD8+ regulatoryT cells. Such cells can be identified by one skilled in the art using,e.g., flow cytometry to identify markers such as FoxP3, LAP or Helios.

As used herein, “regulatory T cells” or “Tregs” refer toimmunosuppressive cells that generally suppress or downregulateinduction and proliferation of effector T cells. Tregs generally expressthe biomarkers CD4, FOXP3, and CD25 and are thought to be derived fromthe same lineage as naïve CD4 cells.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) with cytolytic activities as well as T helper (Th) cells, whichsecrete inflammatory cytokines and activate and direct other immunecells, but does not include regulatory T cells (Treg cells).

As used herein, “administering” refers to the physical introduction of amolecule (e.g., an antibody or antigen binding fragment that binds LAP)or of a composition comprising a therapeutic agent (e.g., an anti-LAPantibody or antigen binding fragment) to a subject, using any of thevarious methods and delivery systems known to those skilled in the art.Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, “cancer” refers to a broad group of diseasescharacterized by the uncontrolled growth of abnormal cells in the body.Unregulated cell division may result in the formation of malignanttumors or cells that invade neighboring tissues and may metastasize todistant parts of the body through the lymphatic system or bloodstream.

As used herein, “autoimmune disease” describes a disease state orsyndrome whereby a subject's body produces a dysfunctional immuneresponse against the subject's own body components, with adverseeffects.

As used herein, “fibrosis” refers to disorders or disease states thatare caused by or accompanied by the abnormal deposition of extracellularmatrix (i.e., not formation of fibrous tissue in normal organ andtissue). Fibrosis is characterized by excessive accumulation ofextracellular matrix in the affected tissue that often results indestruction of its normal architecture and causes significant organdysfunction. Although fibrotic conditions in various organs have diverseetiologies, fibrosis typically results from chronic persistentinflammation induced by a variety of stimuli, such as chronicinfections, ischemia, allergic and autoimmune reactions, chemicalinsults or radiation injury (from Biemacka, 2011 Growth Factors. 2011October; 29(5): 196-202. doi: 10.3109/08977194.2011.595714. Epub 2011Jul. 11). Fibrosis may affect the heart, liver, kidney, lung and skinand is also a central feature in many cancers.

As used herein, “cell therapy” refers to a method of treatment involvingthe administration of live cells (e.g., CAR T cells, and NK cells).

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent (e.g., an anti-LAP antibody or antigen binding fragment)to, the subject with the objective of reversing, alleviating,ameliorating, inhibiting, or slowing down or preventing the progression,development, severity or recurrence of a symptom, complication,condition or biochemical indicia associated with a disease. Treatmentcan be of a subject having a disease or a subject who does not have adisease (e.g., for prophylaxis).

As used herein, “adjunctive” or “combined” administration(co-administration) includes simultaneous administration of the agentsand/or compounds in the same or different dosage form, or separateadministration of the compounds (e.g., sequential administration). Forexample at least one agent comprises an anti-LAP antibody or antigenbinding fragment. Thus, a first antibody or antigen binding fragment,e.g., an anti-LAP antibody or antigen binding fragment, and a second,third, or more antibodies or antigen binding fragments can besimultaneously administered in a single formulation. Alternatively, thefirst and second (or more) antibodies or antigen binding fragments canbe formulated for separate administration and are administeredconcurrently or sequentially.

“Combination” therapy, as used herein, means administration of two ormore therapeutic agents in a coordinated fashion, and includes, but isnot limited to, concurrent dosing. Specifically, combination therapyencompasses both co-administration (e.g. administration of aco-formulation or simultaneous administration of separate therapeuticcompositions) and serial or sequential administration, provided thatadministration of one therapeutic agent is conditioned in some way onadministration of another therapeutic agent. For example, onetherapeutic agent may be administered only after a different therapeuticagent has been administered and allowed to act for a prescribed periodof time. (See, e.g., Kohrt et al. (2011) Blood 117:2423). For example,the anti-LAP antibody can be administered first followed by (e.g.,immediately followed by) the administration of a second antibody (e.g.,an anti-PD-1 antibody) or antigen binding fragment, or vice versa. Inone embodiment, the anti-LAP antibody or antigen binding fragment isadministered prior to administration of the second antibody or antigenbinding fragment. In another embodiment, the anti-LAP antibody orantigen binding fragment is administered, for example, a few minutes(e.g., within about 30 minutes) or at least one hour of the secondantibody or antigen binding fragment. Such concurrent or sequentialadministration preferably results in both antibodies or antigen bindingfragments being simultaneously present in treated patients.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug (e.g., anti-LAP antibody or antigen binding fragment) is anyamount of the drug or therapeutic agent that, when used alone or incombination with another therapeutic agent, promotes disease regressionevidenced by a decrease in severity of disease symptoms, an increase ortherapeutic agent in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. A therapeutically effective amount or dosage of a drug ortherapeutic agent includes a “prophytactically effective amount” or a“prophylactically effective dosage”, which is any amount of the drug ortherapeutic agent that, when administered alone or in combination withanother therapeutic agent to a subject at risk of developing a diseaseor of suffering a recurrence of disease, inhibits the development orrecurrence of the disease. The ability of a therapeutic agent to promotedisease regression or inhibit the development or recurrence of thedisease can be evaluated using a variety of methods known to the skilledpractitioner, such as in human subjects during clinical trials, inanimal model systems predictive of efficacy in humans, or by assayingthe activity of the agent in in vitro assays.

The administration of effective amounts of the anti-LAP antibody orantigen binding fragment alone, or anti-LAP antibody or antigen bindingfragment combined with another compound or agent (e.g., an immunecheckpoint blocker such as an anti-PD-1 antibody), according to any ofthe methods provided herein, can result in at least one therapeuticeffect, including, for example, reduced tumor growth or size, reducednumber of indicia of cancer (e.g., metastatic lesions) appearing overtime, complete remission, partial remission, or stable disease. Forexample, the methods of treatment produce a comparable clinical benefitrate (CBR=complete remission (CR)+partial remission (PR)+stable disease(SD) lasting ≥6 months) better than that achieved without administrationof the anti-LAP antibody or antigen binding fragment, or than thatachieved with administration of any one of the combined antibodies,e.g., the improvement of clinical benefit rate is about 20% 20%, 30%,40%, 50%, 60%, 70%, 80% or more.

By way of example, for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug or therapeutic agent (e.g.,anti-LAP antibody or antigen binding fragment) inhibits tumor cellgrowth by at least about 20%, by at least about 30% by at least about40%, by at least about 50%, by at least about 60%, by at least above70%, by at least about 80%, or by at least about 90% relative tountreated subjects. In some embodiments, a therapeutically effectiveamount or dosage of the drug or therapeutic agent completely inhibitscell growth or tumor growth, i.e., inhibits cell growth or tumor growthby 100%. The ability of a compound or therapeutic agent, including anantibody, to inhibit tumor growth can be evaluated using the assaysdescribed herein. Alternatively, this property of a compositioncomprising the compound or therapeutic agent can be evaluated byexamining the ability of the composition to inhibit cell growth; suchinhibition can be measured in vitro by assays known to the skilledpractitioner.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions described herein canbe used to treat a subject having cancer. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, cats, dogs, cows, chickens, amphibians,reptiles, etc.

The term “sample” refers to tissue, bodily fluid, or a cell (or afraction of any of the foregoing) taken from a patient or a subject.Normally, the tissue or cell will be removed from the patient, but invivo diagnosis is also contemplated. In the case of a solid tumor, atissue sample can be taken from a surgically removed tumor and preparedfor testing by conventional techniques. In the case of lymphomas andleukemias, lymphocytes, leukemic cells, or lymph tissues can be obtained(e.g., leukemic cells from blood) and appropriately prepared. Othersamples, including urine, tears, serum, plasma, cerebrospinal fluid,feces, sputum, cell extracts etc. can also be useful for particularcancers.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising,” “consisting essentially of,” and“consisting of” may be optionally replaced with either of the other twoterms, thus describing alternative aspects of the scope of the subjectmatter. The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. The use of “or”or “and” means “and/or” unless stated otherwise. Furthermore, use of theterm “including” as well as other forms, such as “include,” “includes,”and “included,” is not limiting.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration and the like, encompassesvariations of up to ±10% from the specified value. Unless otherwiseindicated, all numbers expressing quantities of ingredients, propertiessuch as molecular weight, reaction conditions, etc., used herein are tobe understood as being modified by the term “about”.

As used herein, “and/or” is to be taken as specific disclosure of eachof the two specified features or components with or without the other.Thus, the term “and/or” as used in a phrase such as “A and/or B”includes “A and B,” “A or B,” “A” alone, and “B” alone. Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” encompasseseach of the following: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A alone; B alone; and C alone.

As used herein, the terms “ug” and “uM” are used interchangeably with“μg” and “μM,” respectively.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-LAP Antibodies

In one aspect, provided herein is an isolated anti-LAP antibody (i.e.,an antibody that binds LAP) or antigen binding fragment thereof.

In one aspect, provided herein is an isolated anti-LAP antibody (e.g.,recombinant humanized, chimeric, or human antibody) or antigen bindingfragment thereof which comprises:

(a) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 26, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively;

(b) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 27, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively;

(c) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 28, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively;

(d) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 29, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively;

(e) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 30, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively;

(f) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(g) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 64, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(h) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 65, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(i) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(j) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 67, and56, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(k) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and68, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(l) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and69, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(m) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and70, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(n) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and68, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 57, 58, and 59, respectively;

(o) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(p) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(q) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 121, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(r) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 122, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(s) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 123, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(t) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 124, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(u) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 125, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(v) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 126, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively;

(w) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 163, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(x) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 172, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(y) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 173, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(z) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 174, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(aa) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 174, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(ab) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 176, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(ac) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 177, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(ad) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 162, 178, and164, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 165, 166, and 167, respectively;

(ae) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 180, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively; or

(af) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 189, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(ag) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 190, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(ah) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 191, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(ai) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 192, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(aj) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 193, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(ak) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 194, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(al) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 179, 195, and181, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 182, 183, and 184, respectively;

(am) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 225, 226, and227, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 228, 229, and 230, respectively;

(an) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 225, 231, and227, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 228, 229, and 230, respectively;

(ao) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 225, 232, and227, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 228, 229, and 230, respectively; or

(ap) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 225, 233, and227, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 228, 229, and 230, respectively.

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 19,20, and 21, respectively, except wherein position 56 of the heavy chainvariable region (corresponding to position 7 of SEQ ID NO: 17) is anamino acid other than N (e.g., Q, S, H, L, D)) or is substituted with anamino acid residue other than N (e.g., Q, S, H, L, D).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57,58, and 59, respectively, except wherein position 54 of the heavy chainvariable region (corresponding to position 5 of SEQ ID NO: 55) is anamino acid other than N (e.g., Q, A, H, S) or is substituted with anamino acid residue other than N (e.g., Q, A, H, S).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57,58, and 59, respectively, except wherein position 102 of the heavy chainvariable region (corresponding to position 4 of SEQ ID NO: 56) is anamino acid other than D (e.g., A, E, G)) or is substituted with an aminoacid residue other than D (e.g., A, E, G).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57,58, and 59, respectively, except wherein position 54 of the heavy chainvariable region (corresponding to position 5 of SEQ ID NO: 55) is anamino acid other than N (e.g., Q, A, H, S) or is substituted with anamino acid residue other than N (e.g., Q, A, H, S), and wherein position102 of the heavy chain variable region (corresponding to position 4 ofSEQ ID NO: 56) is an amino acid other than D (e.g., A, E, G)) or issubstituted with an amino acid residue other than D (e.g., A, E, G).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 110, 111, and 112,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113,114, and 115, respectively, except wherein position 54 of the heavychain variable region (corresponding to position 5 of SEQ ID NO: 111) isan amino acid other than N (e.g., Q, G, A, S, H, L, D)) or issubstituted with an amino acid residue other than N (e.g., Q, G, A, S,H, L, D).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 162, 163, and 164,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 165,166, and 167, respectively, except wherein position 56 of the heavychain variable region (corresponding to position 7 of SEQ ID NO: 163) isan amino acid other than N (e.g., Q, G, A, S, H, L, D) or is substitutedwith an amino acid residue other than N (Q, G, A, S, H, L, D).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) or antigen binding fragment comprises aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 179, 180, and 181,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 182,183, and 184, respectively, except wherein position 55 of the heavychain variable region (corresponding to position 6 of SEQ ID NO: 180) isan amino acid other than N (e.g., Q, G, A, S, H, L, D) or is substitutedwith an amino acid residue other than N (e.g., Q, G, A, S, H, L, D).

In some embodiments, the anti-LAP antibody (e.g., recombinant humanized,chimeric, or human antibody) comprises a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 225, 226, and 227, respectively, and a lightchain variable region comprising CDR1, CDR2, and CDR3 regions comprisingthe amino acid sequences of SEQ ID NOs: 228, 229, and 230, respectively,except wherein position 55 of the heavy chain variable region(corresponding to position 5 of SEQ ID NO: 226) is an amino acid otherthan D (e.g., G, A, E) or is substituted with an amino acid residueother than D (e.g., G, A, E).

In some embodiments, the anti-LAP antibody comprises the heavy chain CDRsequences of any of subparts (a)-(ap) above, and a constant region,e.g., a human IgG constant region (e.g., IgG1, IgG2, IgG3, or IgG4, orvariants thereof). In some embodiments, the constant region is a humanIgG1 constant region comprising the amino acid sequence set forth in SEQID NO: 196. In some embodiments, the constant region is a variant humanIgG4 constant region comprising the amino acid sequence set forth in SEQID NO: 197. In some embodiments, a heavy chain variable regioncomprising the heavy chain CDR sequences of any of subparts (a)-(ap)above may be linked to a constant domain to form a heavy chain (e.g., afull length heavy chain). Similarly, a light chain variable regioncomprising the light chain CDR sequences of any of subparts (a)-(ap)above may be linked to a constant region to form a light chain (e.g., afull length light chain). A full length heavy chain (with the exceptionof the C-terminal lysine (K) or with the exception of the C-terminalglycine and lysine (GK), which may be absent or removed) and full lengthlight chain combine to form a full length antibody.

In another aspect, provided herein are isolated anti-LAP antibodiescomprising:

(a) heavy and light chain variable region sequences comprising SEQ IDNOs: 42 and 52, respectively;(b) heavy and light chain variable region sequences comprising SEQ IDNOs: 40 and 52, respectively;(c) heavy and light chain variable region sequences comprising SEQ IDNOs: 35 and 46, respectively;(d) heavy and light chain variable region sequences comprising SEQ IDNOs: 35 and 50, respectively;(e) heavy and light chain variable region sequences comprising SEQ IDNOs: 101 and 104, respectively;(f) heavy and light chain variable region sequences comprising SEQ IDNOs: 98 and 104, respectively;(g) heavy and light chain variable region sequences comprising SEQ IDNOs: 92 and 104, respectively;(h) heavy and light chain variable region sequences comprising SEQ IDNOs: 92 and 106, respectively;(i) heavy and light chain variable region sequences comprising SEQ IDNOs: 95 and 104, respectively;(j) heavy and light chain variable region sequences comprising SEQ IDNOs: 77 and 104, respectively;(k) heavy and light chain variable region sequences comprising SEQ IDNOs: 82 and 104, respectively;(l) heavy and light chain variable region sequences comprising SEQ IDNOs: 87 and 104, respectively;(m) heavy and light chain variable region sequences comprising SEQ IDNOs: 133 and 154, respectively;(n) heavy and light chain variable region sequences comprising SEQ IDNOs: 130 and 154, respectively;(o) heavy and light chain variable region sequences comprising SEQ IDNOs: 127 and 154, respectively;(p) heavy and light chain variable region sequences comprising SEQ IDNOs: 144 and 154, respectively;(q) heavy and light chain variable region sequences comprising SEQ IDNOs: 146 and 154, respectively;(r) heavy and light chain variable region sequences comprising SEQ IDNOs: 148 and 154, respectively;(s) heavy and light chain variable region sequences comprising SEQ IDNOs: 150 and 154, respectively; or(t) heavy and light chain variable region sequences comprising SEQ IDNOs: 218 and 154, respectively.

In some embodiments, the anti-LAP antibody has variable region sequenceswith potential liability sites, e.g., deamidation sites and/orisomerization sites) removed.

Accordingly, in some embodiments, the anti-LAP antibody comprises heavyand light chain variable region sequences of any of subparts (a)-(d)above, except wherein position 56 of the heavy chain variable region isan amino acid other than N (e.g., Q, S, H, L, D)) or is substituted withan amino acid residue other than N (e.g., Q, S, H, L, D).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain variable region sequences of any of subparts (e)-(l) above, exceptwherein position 54 of the heavy chain variable region is an amino acidother than N (e.g., Q, A, H, S) or is substituted with an amino acidresidue other than N (e.g., Q, A, H, S).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain variable region sequences of any of subparts (e)-(l) above, exceptwherein position 102 of the heavy chain variable region is an amino acidother than D (e.g., A, E, G) or is substituted with an amino acidresidue other than D (e.g., A, E, G).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain variable region sequences of any of subparts (e)-(l) above, exceptwherein position 54 of the heavy chain variable region is an amino acidother than N (e.g., Q, A, H, S) or is substituted with an amino acidresidue other than N (e.g., Q, A, H, S), and wherein position 102 of theheavy chain variable region is an amino acid other than D (e.g., A, E,G) or is substituted with an amino acid residue other than D (e.g., A,E, G).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain variable region sequences of any of subparts (m)-(t) above, exceptwherein position 54 of the heavy chain variable region is an amino acidother than N (e.g., Q, G, A, S, H, L, D)) or is substituted with anamino acid residue other than N (e.g., Q, G, A, S, H, L, D).

In some embodiments, the anti-LAP antibody comprises a heavy chainand/or light chain variable region sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain and/orlight chain variable region sequences of any of subparts (a)-(t) above.

In some embodiments, the heavy chain and/or light chain variable regionsequences of any of subparts (a)-(t) above has 1, 2, 3, 4, 5, 1-2, 1-3,1-4, or 1-5 amino acid substitutions (e.g., conservative amino acidsubstitutions). In some embodiments, the anti-LAP antibody does not haveheavy and light chain variable region sequences which are identical toSEQ ID NOs: 22 and 23, respectively; 60 and 61, respectively; 116 and117, respectively; 168 and 169, respectively; or 185 and 186,respectively. These anti-LAP antibodies can be tested for variousproperties that are clinically advantageous (e.g., binding to LAP-TGFβ1,inhibiting the activation of TGFβ1, binding to various cell (e.g.,immune cell) populations, inhibiting tumor growth in vivo) using theassays and animal models described herein, for example, in the Examples.

In some embodiments, the anti-LAP antibody comprises the heavy chainvariable region sequences of any of subparts (a)-(t) above, and aconstant region, e.g., a human IgG constant region (e.g., IgG1, IgG2,IgG3, or IgG4, or variants thereof). In some embodiments, the constantregion is a human IgG1 constant region comprising the amino acidsequence set forth in SEQ ID NO: 196. In some embodiments, the constantregion is a variant human IgG4 constant region comprising the amino acidsequence set forth in SEQ ID NO: 197. In some embodiments, the heavychain variable region sequences of any of subparts (a)-(t) above may belinked to a constant domain to form a heavy chain (e.g., a full lengthheavy chain). Similarly, the light chain variable region sequences ofany of subparts (a)-(t) above may be linked to a constant region to forma light chain (e.g., a full length light chain). A full length heavychain (with the exception of the C-terminal lysine (K) or with theexception of the C-terminal glycine and lysine (GK), which may be absentor removed) and full length light chain combine to form a full lengthantibody.

Accordingly, in some embodiments, provided herein are anti-LAPantibodies comprising (a) heavy chain CDR1-3 sequences comprising SEQ IDNOs: 225, 231, and 227, respectively, and light chain CDR1-3 sequencescomprising SEQ ID NOs: 228, 229, and 230, respectively, (b) heavy chainCDR1-3 sequences comprising SEQ ID NOs: 225, 232, and 227, respectively,and light chain CDR1-3 sequences comprising SEQ ID NOs: 228, 229, and230, respectively, or (c) heavy chain CDR1-3 sequences comprising SEQ IDNOs: 225, 233, and 227, respectively, and light chain CDR1-3 sequencescomprising SEQ ID NOs: 228, 229, and 230, respectively.

In some embodiments, provided herein are antibodies comprising heavy andlight chain variable region sequences comprising (a) SEQ ID NOs: 234 and224, respectively, (b) SEQ ID NOs: 235 and 224, respectively, or (c) SEQID NOs: 236 and 224, respectively.

In some embodiments, provided herein are anti-LAP antibodies comprisingheavy and light chain sequences comprising (a) SEQ ID NOs: 237 and 222,respectively, (b) SEQ ID NOs: 238 and 222, respectively, or (c) SEQ IDNOs: 239 and 222, respectively.

In another aspect, provided herein are isolated anti-LAP antibodiescomprising:

(a) heavy and light chain sequences comprising SEQ ID NOs: 43 and 53,respectively;(b) heavy and light chain sequences comprising SEQ ID NOs: 45 and 53,respectively;(c) heavy and light chain sequences comprising SEQ ID NOs: 41 and 53,respectively;(d) heavy and light chain sequences comprising SEQ ID NOs: 36 and 47,respectively;(e) heavy and light chain sequences comprising SEQ ID NOs: 37 and 47,respectively;(f) heavy and light chain sequences comprising SEQ ID NOs: 36 and 51,respectively;(g) heavy and light chain sequences comprising SEQ ID NOs: 37 and 51,respectively;(h) heavy and light chain sequences comprising SEQ ID NOs: 102 and 105,respectively;(i) heavy and light chain sequences comprising SEQ ID NOs: 103 and 105,respectively;(j) heavy and light chain sequences comprising SEQ ID NOs: 99 and 105,respectively;(k) heavy and light chain sequences comprising SEQ ID NOs: 100 and 105,respectively;(l) heavy and light chain sequences comprising SEQ ID NOs: 93 and 105,respectively;(m) heavy and light chain sequences comprising SEQ ID NOs: 94 and 105,respectively;(n) heavy and light chain sequences comprising SEQ ID NOs: 93 and 107,respectively;(o) heavy and light chain sequences comprising SEQ ID NOs: 94 and 107,respectively;(p) heavy and light chain sequences comprising SEQ ID NOs: 96 and 105,respectively;(q) heavy and light chain sequences comprising SEQ ID NOs: 97 and 105,respectively;(r) heavy and light chain sequences comprising SEQ ID NOs: 78 and 105,respectively;(s) heavy and light chain sequences comprising SEQ ID NOs: 79 and 105,respectively;(t) heavy and light chain sequences comprising SEQ ID NOs: 83 and 105,respectively;(u) heavy and light chain sequences comprising SEQ ID NOs: 84 and 105,respectively;(v) heavy and light chain sequences comprising SEQ ID NOs: 88 and 105,respectively;(w) heavy and light chain sequences comprising SEQ ID NOs: 89 and 105,respectively;(x) heavy and light chain sequences comprising SEQ ID NOs: 134 and 155,respectively;(y) heavy and light chain sequences comprising SEQ ID NOs: 135 and 155,respectively;(z) heavy and light chain sequences comprising SEQ ID NOs: 131 and 155,respectively;(aa) heavy and light chain sequences comprising SEQ ID NOs: 132 and 155,respectively;(ab) heavy and light chain sequences comprising SEQ ID NOs: 128 and 155,respectively;(ac) heavy and light chain sequences comprising SEQ ID NOs: 129 and 155,respectively;(ad) heavy and light chain sequences comprising SEQ ID NOs: 145 and 155,respectively;(ae) heavy and light chain sequences comprising SEQ ID NOs: 147 and 155,respectively;(af) heavy and light chain sequences comprising SEQ ID NOs: 149 and 155,respectively;(ag) heavy and light chain sequences comprising SEQ ID NOs: 151 and 155,respectively;(ah) heavy and light chain sequences comprising SEQ ID NOs: 219 and 155,respectively; or(ai) heavy and light chain sequences comprising SEQ ID NOs: 220 and 155,respectively.

In some embodiments, the full length heavy chain lacks the C-terminallysine residue (which may be absent or removed).

In some embodiments, the anti-LAP antibody has heavy and light chainsequences with potential liability sites, e.g., deamidation sites and/orisomerization sites) removed.

Accordingly, in some embodiments, the anti-LAP antibody comprises heavyand light chain sequences of any of subparts (a)-(g) above, exceptwherein position 56 of the heavy chain is an amino acid other than N(e.g., Q, S, H, L, D) or is substituted with an amino acid residue otherthan N (e.g., Q, S, H, L, D).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain sequences of any of subparts (h)-(w) above, except whereinposition 54 of the heavy chain is an amino acid other than N (e.g., Q,A, H, S) or is substituted with an amino acid residue other than N, H,or S (e.g., Q, A, H, S).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain sequences of any of subparts (h)-(w) above, except whereinposition 102 of the heavy chain variable region is an amino acid otherthan D (e.g., A, E, G) or is substituted with an amino acid residueother than D (e.g., A, E, G).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain sequences of any of subparts (h)-(w) above, except whereinposition 54 of the heavy chain is an amino acid other than N (e.g., Q,A, H, S) or is substituted with an amino acid residue other than N, H,or S (e.g., Q, A, H, S) and position 102 of the heavy chain variableregion is an amino acid other than D (e.g., A, E, G) or is substitutedwith an amino acid residue other than D (e.g., A, E, G).

In some embodiments, the anti-LAP antibody comprises heavy and lightchain sequences of any of subparts (x)-(ai) above, except whereinposition 54 of the heavy chain variable region is an amino acid otherthan N (e.g., Q, G, A, S, H, L, D) or is substituted with an amino acidresidue other than N (e.g., Q, G, A, S, H, L, D).

In some embodiments, the anti-LAP antibody comprises a heavy chainand/or light chain sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5%, or 99.7% identical to the heavy chain and/or lightchain sequences of any of subparts (a)-(ai) above. In some embodiments,the heavy chain and/or light chain sequences of any of subparts (a)-(ai)above has 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acid substitutions(e.g., conservative amino acid substitutions). In some embodiments, theanti-LAP antibody does not have a heavy and/or light chain variableregion sequence which is identical to SEQ ID NOs: 24 and 25,respectively; 62 and 63 respectively; or 118 and 119, respectively.These anti-LAP antibodies can be tested for various properties that areclinically advantageous (e.g., binding to LAP-TGFβ1, inhibiting theactivation of TGFβ1, binding to various cell (e.g., immune cell)populations, inhibiting tumor growth in vivo) using the assays andanimal models described herein, for example, in the Examples.

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprising VHCDR1-3 sequences of SEQ ID NOs: 110, 120, and 113,respectively, and VLCDR1-3 sequences of SEQ ID NOs: 113, 114, and 115,respectively, has one or more amino acid substitutions in the CDRs orvariable regions. For example, in some embodiments, no more than 3 aminoacids (i.e., 1, 2, or 3 amino acids) are substituted in the six heavyand light chain CDRs (collectively), or two heavy and light chainvariable regions (collectively).

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VHCDR1 which has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, for example, conservative amino acid substitutions,relative to GYTFTSYWMH (SEQ ID NO: 110).

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VHCDR2 which has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, for example, conservative amino acid substitutions,relative to RIDPQSGGIK (SEQ ID NO: 120). In some embodiments, the VHCDR2comprises the sequence: RX₁X₂X₃X₄X₅X₆X₇X₈X₉, wherein X₁-X₉; can be anyamino acid. In some embodiments, only 1 position among X₁-X₃; issubstituted relative to the amino acid sequence of SEQ ID NO: 120.

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VHCDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions, for example, conservative amino acid substitutions,relative to WDYGGYFDV (SEQ ID NO: 112). In some embodiments, the VHCDR3comprises the sequence: WX₁YGGYFX₂X₃ (SEQ ID NO: 242), wherein X₁-X₃ canbe any amino acid. In some embodiments, only 1 position among X₁-X₃ issubstituted relative to the amino acid sequence of SEQ ID NO: 112.

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VLCDR1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 aminoacid substitutions, for example, conservative amino acid substitutions,relative to RASQDITNYLN (SEQ ID NO: 113). In some embodiments, theVLCDR1 may comprise the sequence: RX₁X₂X₃DIX₄X₅YX₆X₇, wherein X₁-X₇ isany amino acid. In some embodiments, only 1 position among X₁-X₇ issubstituted relative to the amino acid sequence of SEQ ID NO: 113.

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VLCDR2 comprising 1, 2, 3, 4, 5, 6, or 7 amino acidsubstitutions, for example, conservative amino acid substitutions,relative to YTSRLHS (SEQ ID NO: 114). In some embodiments, the VLCDR2comprises the sequence: YX₁X₂RX₃X₄X₅, wherein X₁-X₅ is any amino acid.In some embodiments, only 1 position among X₁-X₅ is substituted relativeto the amino acid sequence of SEQ ID NO: 114.

In some embodiments, an anti-LAP antibody or antigen binding fragmentcomprises a VLCDR3 comprising 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsubstitutions, for example, conservative amino acid substitutions,relative to QQGDTLPWT (SEQ ID NO: 115). In some embodiments, the VLCDR3may comprise the sequence: QQGDXiLPWT (SEQ ID NO: 243), wherein X₁ isany amino acid.

Functional features of the anti-LAP antibodies or antigen bindingfragment provided herein are described below in more detail.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 (e.g., human LAP-TGFβ1) in theabsence of an anchor protein. For example, the anti-LAP antibody orantigen binding fragment described herein binds to recombinant humanLAP-TGFβ1 in an assay that does not include an anchor protein.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 (e.g., soluble LAP-TGFβ1) with aK_(D) of 100 nM or less, such as 90 nM or less, 80 nM or less, 70 nM orless, 60 nM or less, 50 nM or less, such as 40 nM or less, 30 nM orless, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 1 nM orless, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less,0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nMor less, 10 nM to 0.1 nM, 5 nM to 0.1 nM, 3 nM to 0.1 nM, 1 nM to 0.1nM, 0.8 nM to 0.1 nM, 0.5 nM to 0.1 nM, 10 nM to 0.5 nM, 10 nM to 0.8nM, 10 nM to 1 nM, 1 nM to 0.5 nM, or 1 nM to 0.8 nM, as assessed by,e.g., bio-layer interferometry (e.g., as described in Example 1), or asdetermined by Octet or BIACore. In some embodiments, the anti-LAPantibody or antigen binding fragment described herein binds to LAP-TGFβ1(e.g., human, cyno, rat and) with a K_(D) in an Example herein. Invarious embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to human LAP-TGFβ1, rat LAP-TGFβ1, cynoLAP-TGFβ1, and/or murine LAP-TGFβ1.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein described herein binds to LAP-TGFβ1 complexed with ananchor protein on immunosuppressive cells, but does not bind to theanchor protein. In some embodiments, the anchor protein is GARP orLRRC33.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein described herein selectively inhibits TGFβ1 activationon immunosuppressive cells without inhibiting TGFβ1 activation onextracellular matrix.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein does not bind to LAP complexed with LTBP1, LTBP3,and/or LTBP4.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein does not bind to LAP-TGFβ2 (e.g., human LAP-TGFβ2) andLAP-TGFβ3 (e.g., human LAP-TGFβ3), as assessed by, e.g., flow cytometryusing cells that overexpress TGFβ2 or TGFβ3, or bio-layer interferometrywith recombinant LAP-TGFβ2 or LAP-TGFβ3. For example, in someembodiments, the anti-LAP antibody or antigen binding fragment describedherein binds to LAP-TGFβ2 or LAP-TGFβ3 with a signal or affinity that isnot significantly above the signal seen with a control antibody (e.g.,isotype control) or the signal seen in the absence of anti-LAP antibody(e.g., as described in Example 2).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein inhibits TGFβ1 activation, as assessed by, e.g., ELISAdetection of free TGFβ1 in a culture of P3U1 cells overexpressingLAP-TGFβ1. In some embodiments, the anti-LAP antibody or antigen bindingfragment described herein inhibits (or is determined to inhibit) TGFβ1activation by about 50% or more, e.g., by about 60% or more, by about70% or more, by about 80% or more, or by about 90% or more, as assessedby ELISA, e.g., ELISA detection of free TGFβ1 in a culture of P3U1 cellsoverexpressing LAP-TGFβ1 (e.g., as described in Example 4).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to mouse and human LAP-TGFβ1, as assessed by,e.g., flow cytometry of activated immune cell populations.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein does not bind to free TGFβ1 (i.e., TGFβ1 without LAP),as assessed by, e.g., ELISA. In some embodiments, the anti-LAP antibodyor antigen binding fragment described herein does not bind to empty LAP(i.e., LAP that is not complexed with TGFβ1), as assessed by, e.g.,bio-layer interferometry. For example, in some embodiments, the anti-LAPantibody or antigen binding fragment described herein binds to freeTGFβ1 or empty with a signal or affinity that is not significantly abovethe signal seen with a control antibody (e.g., isotype control) or thesignal seen in the absence of anti-LAP antibody (e.g., as described inExample 2).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to human LAP-TGFβ1 comprising K27C and Y75Cmutations (SEQ ID NO: 12. In another embodiment, the anti-LAP antibodyor antigen binding fragment described herein does not bind to (or aredetermined not to bind to) human LAP-TGFβ1 comprising a Y74T mutation(SEQ ID NO: 13). In another embodiment, the anti-LAP antibody or antigenbinding fragment described herein binds to (or is determined to bind to)human LAP-TGFβ1 comprising K27C and Y75C mutations, but not to LAP-TGFβ1comprising a Y74T mutation.

In some embodiments, the anti-LAP antibodies bind to all or a portion ofresidues 82-130 of human LAP-TGFβ1 (SEQ ID NO: 1).

In some embodiments, the anti-LAP antibodies bind within residues 82-130of human LAP-TGFβ1 (SEQ ID NO: 1). In some embodiments, the anti-LAPantibody or antigen binding fragment binds to one or more regions onhuman LAP-TGFβ1 (SEQ ID NO: 1) comprising or consisting of amino acids31-40, 274-280, and 340-343. In some embodiments, the anti-LAP antibodyor antigen binding fragment binds to amino acids 31-40, 274-280, and340-343 of human LAP-TGFβ1 (SEQ ID NO: 1). In some embodiments, theepitope is determined by cryo-EM.

In some embodiments, the anti-LAP antibody or antigen binding fragmentbinds to one or more regions on human an LAP-TGFβ1 (SEQ ID NO: 1)comprising or consisting of amino acids 31-38, 278-281, and 342-344. Insome embodiments, the anti-LAP antibodies bind to amino acids 31-38,278-281, and 342-344 of human LAP-TGFβ1 (SEQ ID NO: 1). In someembodiments, the epitope is determined by cryo-EM. In some embodiments,the anti-LAP antibody or antigen binding fragment binds to one or moreregions on human an LAP-TGFβ1 (SEQ ID NO: 1) comprising or consisting ofamino acids 35-43, 272-275, 280-283, and 340 (SEQ ID NO: 1). In someembodiments, the anti-LAP antibody or antigen binding fragment binds toamino acids 35-43, 272-275, 280-283, and 340 of human LAP-TGFβ1 (SEQ IDNO: 1). In some embodiments, the epitope is determined by cryo-EM.

As discussed above, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 on cells, such as immune cells,e.g., immunosuppressive cells. Immunosuppressive cells include, but arenot limited to, suppressive T cells (e.g., regulatory T cells, activatedT cells, suppressive CD8+ T cells), M1 macrophages, M2 macrophages,dendritic cells, regulatory B cells, granulocytic MDSCs, and/ormonocytic MDSCs, as assessed, e.g., by flow cytometry. In someembodiments, the anti-LAP antibody or antigen binding fragment describedherein binds to cells other than immune cells, such as tumor cells,fibroblasts (including cancer associated fibroblasts), mesenchymalstromal cells, mesenchymal stem cells, hemopoietic stem cells,non-myelinating Schwann cells, myofibroblasts, endothelial cells,platelets, megakaryocytes, pericytes, and/or hepatic stellate cells. Insome embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 on both immune cells (e.g.,immunosuppressive cells) and non-immune cells.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 on GARP-positive cells (e.g.,GARP-positive immunosuppressive cells). In some embodiments, theanti-LAP antibody or antigen binding fragment described herein binds to(or are determined to bind to) LAP-TGFβ1 on GARP-negative cells (e.g.,GARP-negative immunosuppressive cells). In some embodiments, theanti-LAP antibody or antigen binding fragment described herein binds toLAP-TGFβ1 on both GARP-positive and GARP-negative cells, as assessed,e.g., by flow cytometry.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein reduces the endogenous expression of CD73. In someembodiments, the anti-LAP antibody or antigen binding fragment describedherein inhibits the increase of CD73 expression caused by a treatment,e.g., radiation. CD73 expression can be determined using standardmethods known in the art (e.g., as described in Example 16).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 expressed on cells (e.g., human ormouse LAP-TGFβ1 expressed on, e.g., P3U1 cells) with an EC₅₀ of 1000ng/ml or less, 500 ng/ml or less, 200 ng/ml or less, 150 ng/ml or less,100 ng/ml or less, 50 ng/ml or less, 25 ng/ml or less, 10 ng/ml or less,5 ng/ml or less, 2 ng/ml or less, 1 ng/ml to 200 ng/ml, 1 ng/ml to 150ng/ml, 1 ng/ml to 100 ng/ml, 1 ng/ml to 50 ng/ml, 1 ng/ml to 25 ng/ml, 1ng/ml to 10 ng/ml, or 1 ng/ml to 5 ng/ml, as measured by flow cytometry(e.g., as described in Example 2).

The binding of the anti-LAP antibody or antigen binding fragment toLAP-TGFβ1 may also be defined using quantitative immunofluorescence byflow cytometry, which allows the number of antibody molecules bound percell to be quantified. Accordingly, in some embodiments, the number ofanti-LAP antibodies bound to a cell that also expresses GARP may beequal to the number of anti-GARP antibodies bound to that cell, or maybe at least 80%, at least 50%, at least 20%, at least 10%, at least 5%,at least 1%, or at least 0.1% of the number of anti-GARP antibodiesbound to that cell. In some embodiments, the number of LAP-TGFβ1molecules expressed per cell may be quantified using quantitativeimmunofluorescence using an anti-LAP antibody of a group that detectsthe majority of LAP molecules; examples of such antibodies include 2F8,2C9, 16B4 and the anti-LAP monoclonal antibody #27232 (R&D Systems). Insome embodiments, the number of anti-LAP antibodies bound to the cellmay be equal to the number of LAP molecules on the cell, or may be atleast 80%, at least 50%, at least 20%, at least 10%, at least 5%, atleast 1% or at least 0.1% of the number of LAP molecules expressed onthat cell.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein inhibits TGFβ1 activation by, for example, 10% or more,for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, 90% or more, or 95% or more, relative toa control (e.g., a control antibody), as measured by ELISA (e.g., asdescribed in Example 4).

Preferably, the anti-LAP antibody or antigen binding fragment describedherein binds to soluble LAP-TGFβ1 with high affinity, for example, witha K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M orless, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, or 10⁻⁹ M to 10⁻⁷ M, as measured by bio-layerinterferometry (e.g., as described in Example 1).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein does not bind to LAP-TGFβ1 in the extracellular matrix.For example, the anti-LAP antibody or antigen binding fragment describedherein do not bind to LAP-TGFβ1 in the extracellular matrix, as assessedby ELISA, wherein the O.D. signal for the antibody or antigen bindingfragment binding is not significantly above the signal seen in theabsence of the anti-LAP antibody or antigen binding fragment describedherein or the signal seen with a control antibody (e.g., isotypecontrol) (e.g., as described in Example 5).

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein do not inhibit TGFβ activation in the ECM, as assessedby, e.g., ELISA detection of free TGFβ1 in an assay combining a sourceof LAP-TGFβ1 in the ECM (e.g., as described in Example 5) with MMP-2,MMP-9, thrombospondin or cells expressing αVβ6 or αVβ8 integrins.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to LAP-TGFβ1 on platelets. For example, in someembodiments, at least 5%, at least 10%, at least 20% or at least 50% ofplatelets can be detected by binding of the anti-LAP antibody (e.g.display a signal above that seen with an isotype control antibody) byflow cytometry (e.g., as described in Example 6). In some embodiments,the anti-LAP antibody or antigen binding fragment described herein bindsto platelets but do not cause platelet aggregation or plateletdegranulation.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to immune cells, e.g., suppressive T cells (e.g.,regulatory T cells), M2 macrophages, monocytic MDSCs, CD11b-positivecells, and/or dendritic cells. For example, in some embodiments, atleast 0.5%, at least 1%, at least 2%, at least 5%, at least 7%, at least10%, at least 20%, or at least 50% of these cell types can be detectedby binding of the anti-LAP antibody (e.g. display a signal above thatseen with an isotype control antibody) by flow cytometry (e.g., asdescribed in Example 7). In some embodiments, the anti-LAP antibody orantigen binding fragment described herein is considered to bind to thesecell types if they bind ≥2 standard deviations above isotype control.

In some embodiments, the anti-LAP antibody or antigen binding fragmentdescribed herein binds to GARP-negative leukocytes. For example, in someembodiments, at least 0.5%, at least 1%, at least 2%, at least 5%, atleast 7%, at least 10%, at least 20% or at least 50% of GARP-negativeleukocytes can be detected by binding of the anti-LAP antibody (e.g.display a signal above that seen with an isotype control antibody) byflow cytometry (e.g., as described in Example 7).

An antibody or antigen binding fragment that exhibits one or more of thefunctional properties described above (e.g., biochemical,immunochemical, cellular, physiological or other biological activities),as determined using methods known to the art and described herein, willbe understood to relate to a statistically significant difference in theparticular activity relative to that seen in the absence of the antibody(e.g., or when a control antibody of irrelevant specificity is present).Preferably, the anti-LAP antibody-induced increases in a measuredparameter effects a statistically significant increase by at least 10%of the measured parameter, more preferably by at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2 fold), 3 fold, 5 fold or10 fold. Conversely, anti-LAP antibody-induced decreases in a measuredparameter (e.g., TGFβ1 activation) effects a statistically significantdecrease by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97%, 98%, 99%, or 100%.

Also provided herein are anti-LAP antibodies that bind to the sameepitope on human LAP-TGFβ1 as any of the anti-LAP antibodies describedherein. These antibodies have the ability to cross-compete for bindingto human LAP-TGFβ1 with any of the anti-LAP antibodies described herein.In some embodiments, the anti-LAP antibodies bind one or more aminoacids within residues 82-130 of human LAP-TGFβ1 (SEQ ID NO: 1).

Antibodies disclosed herein include all known forms of antibodies andother protein scaffolds with antibody-like properties. For example, theantibody can be a human antibody, a humanized antibody, a bispecificantibody, an immunoconjugate, a chimeric antibody, or a protein scaffoldwith antibody-like properties, such as fibronectin or ankyrin repeats.

In some embodiments, the antibody is a bispecific antibody comprising afirst and second binding region, wherein the first binding regioncomprises the binding specificity (e.g., antigen-binding region) of ananti-LAP antibody described herein, and a second binding region thatdoes not bind to LAP. In some embodiments, the second binding regionbinds to a protein that is not expressed on platelets.

The antibody also can be a Fab, F(ab′)₂, scFv, AFFIBODY, avimer,nanobody, single chain antibody, or a domain antibody. The antibody alsocan have any isotype, including any of the following isotypes: IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Full-lengthantibodies can be prepared from V_(H) and V_(L) sequences using standardrecombinant DNA techniques and nucleic acid encoding the desiredconstant region sequences to be operatively linked to the variableregion sequences.

In certain embodiments, the antibodies described herein may haveeffector function or may have reduced or no effector function. Incertain embodiments, anti-LAP antibodies comprise an effector-less ormostly effector-less Fc, e.g., IgG2 or IgG4. Generally, variable regionsdescribed herein may be linked to an Fc comprising one or moremodification, typically to alter one or more functional properties ofthe antibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Furthermore, anantibody described herein may be chemically modified (e.g., one or morechemical moieties can be attached to the antibody) or be modified toalter its glycosylation, to alter one or more functional properties ofthe antibody. Each of these embodiments is described in further detailbelow. The numbering of residues in the Fc region is that of the EUindex of Kabat.

In some embodiments, the Fc region is a variant Fc region, e.g., an Fcsequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fc sequence (e.g., anunmodified Fc polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity. For example, modifications can be made in the Fc region inorder to generate an Fc variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for Clq and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region may includetwo, three, four, five, etc. substitutions therein, e.g. of the specificFc region positions identified herein.

A variant Fc region may also comprise a sequence alteration whereinamino acids involved in disulfide bond formation are removed or replacedwith other amino acids. Such removal may avoid reaction with othercysteine-containing proteins present in the host cell used to producethe antibodies described herein. Even when cysteine residues areremoved, single chain Fc domains can still form a dimeric Fc domain thatis held together non-covalently. In other embodiments, the Fc region maybe modified to make it more compatible with a selected host cell. Forexample, one may remove the PA sequence near the N-terminus of a typicalnative Fc region, which may be recognized by a digestive enzyme in E.coli such as proline iminopeptidase. In other embodiments, one or moreglycosylation sites within the Fc domain may be removed. Residues thatare typically glycosylated (e.g., asparagine) may confer cytolyticresponse. Such residues may be deleted or substituted withunglycosylated residues (e.g., alanine). In other embodiments, sitesinvolved in interaction with complement, such as the Clq binding site,may be removed from the Fc region. For example, one may delete orsubstitute the EKK sequence of human IgG1. In certain embodiments, sitesthat affect binding to Fc receptors may be removed, preferably sitesother than salvage receptor binding sites. In other embodiments, an Fcregion may be modified to remove an ADCC site. ADCC sites are known inthe art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) withregard to ADCC sites in IgG1. Specific examples of variant Fc domainsare disclosed for example, in PCT Publication numbers WO 97/34631 and WO96/32478.

In one embodiment, the hinge region of Fc is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. More specifically, one or more amino acidmutations are introduced into the CH2-CH3 domain interface region of theFc-hinge fragment such that the antibody has impaired Staphylococcylprotein A (SpA) binding relative to native Fc-hinge domain SpA binding.This approach is described in further detail in U.S. Pat. No. 6,165,745by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal. In another example, one or more amino acid residues within aminoacid positions 231 and 239 are altered to thereby alter the ability ofthe antibody to fix complement. This approach is described further inPCT Publication number WO 94/29351 by Bodmer et al.

In yet another example, the Fc region may be modified to increaseantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity for an Fey receptor by modifying one or more amino acids at thefollowing positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245,247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322,324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434,435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D,239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variantsinclude 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F,267E/324T, and 267E/268F/324T. Other modifications for enhancing FcγRand complement interactions include but are not limited to substitutions298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L,292P, 300L, 396L, 3051, and 396L. These and other modifications arereviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to an Fey receptor include aminoacid modifications at any one or more of amino acid positions 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279,280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373,376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat (PCT Patent Publicationnumber WO00/42072).

Other Fc modifications that can be made to Fes are those for reducing orablating binding to FcγR and/or complement proteins, thereby reducing orablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.Exemplary modifications include but are not limited substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, and 328, wherein numbering is according to the EU index. Exemplarysubstitutions include but are not limited to 234G, 235G, 236R, 237K,267R, 269R, 325L, and 328R, wherein numbering is according to the EUindex. An Fc variant may comprise 236R/328R. Other modifications forreducing FcγR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S,229S, 238S, 233P, and 234V, as well as removal of the glycosylation atposition 297 by mutational or enzymatic means or by production inorganisms such as bacteria that do not glycosylate proteins. These andother modifications are reviewed in Strohl, 2009, Current Opinion inBiotechnology 20:685-691. Optionally, the Fc region may comprise anon-naturally occurring amino acid residue at additional and/oralternative positions known to one skilled in the art (see, e.g., U.S.Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091;8,101,720; PCT Patent Publication numbers WO 00/42072; WO 01/58957; WO02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO06/020114).

Fc variants that enhance affinity for an inhibitory receptor FcγR11b mayalso be used. Such variants may provide an Fc fusion protein withimmunomodulatory activities related to FcγR11b⁺ cells, including forexample B cells and monocytes. In one embodiment, the Fc variantsprovide selectively enhanced affinity to FcγR11b relative to one or moreactivating receptors. Modifications for altering binding to FcγR11binclude one or more modifications at a position selected from the groupconsisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327,328, and 332, according to the EU index. Exemplary substitutions forenhancing FcγR11b affinity include but are not limited to 234D, 234E,234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E,266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D,268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding toFcγR11b include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D,267E/268E, and 267E/328F.

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thismay be done by increasing the binding affinity of the Fc region forFcRn. For example, one or more of more of following residues can bemutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No.6,277,375. Specific exemplary substitutions include one or more of thefollowing: T252L, T254S, and/or T256F. Alternatively, to increase thebiological half-life, the antibody can be altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplaryvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, 428, and 434,including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y,and 434M. Other variants that increase Fc binding to FcRn include: 250E,250Q, 428L, 428F, 250Q/428L (Hinton et al, 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A,272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A(Shields et al, Journal of Biological Chemistry, 2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E,256D, 256T, 309P, 31 1 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dali Acqua et al.Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671. In certain embodiments, hybrid IgG isotypes withparticular biological characteristics may be used. For example, anIgG1/IgG3 hybrid variant may be constructed by substituting IgG1positions in the CH2 and/or CH3 region with the amino acids from IgG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutions,e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R,and 436F. In other embodiments described herein, an IgG1/IgG2 hybridvariant may be constructed by substituting IgG2 positions in the CH2and/or CH3 region with amino acids from IgG1 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutions, e.g., one or more of thefollowing amino acid substitutions: 233E, 234L, 235L, −236G (referringto an insertion of a glycine at position 236), and 327A.

Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which hasbeen shown to exhibit enhanced FcγRIIIa binding and ADCC activity(Shields et al, 2001). Other IgG1 variants with strongly enhancedbinding to FcγRIIIa have been identified, including variants withS239D/I332E and S239D/I332E/A330L mutations which showed the greatestincrease in affinity for FcγRIIIa, a decrease in FcγRIIb binding, andstrong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006).Introduction of the triple mutations into antibodies such as alemtuzumab(CD52-specific), trastuzumab (HER2/neu-specific), rituximab(CD20-specific), and cetuximab (EGFR-specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). Inaddition, IgG1 mutants containing L235V, F243L, R292P, Y300L and P396Lmutations which exhibited enhanced binding to FcγRIIIa and concomitantlyenhanced ADCC activity in transgenic mice expressing human FcγRIIIa inmodels of B cell malignancies and breast cancer have been identified(Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutantsthat may be used include: S298A/E333A/L334A, S239D/I332E,S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.

When using an IgG4 constant domain, it is usually preferable to includethe substitution S228P, which mimics the hinge sequence in IgG1 andthereby stabilizes IgG4 molecules.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al. Glycosylation of the constant region on N297 may be preventedby mutating the N297 residue to another residue, e.g., N297A, and/or bymutating an adjacent amino acid, e.g., 298 to thereby reduceglycosylation on N297.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies described herein to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hanai et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication number WO 03/035835by Presta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication number WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Another modification of the antibodies described herein is pegylation.An antibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C1-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be pegylatedis an aglycosylated antibody. Methods for pegylating proteins are knownin the art and can be applied to the antibodies described herein. Seefor example, European patent number EP 0 154 316 by Nishimura et al. andEuropean patent number EP 0 401 384 by Ishikawa et al.

The affinities and binding properties of an Fc region for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art including, but not limitedto, equilibrium methods (e.g., enzyme-linked immunosorbent assay(ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACOREanalysis), and other methods such as indirect binding assays,competitive inhibition assays, fluorescence resonance energy transfer(FRET), gel electrophoresis, and chromatography (e.g., gel filtration).These and other methods may utilize a label on one or more of thecomponents being examined and/or employ a variety of detection methodsincluding but not limited to chromogenic, fluorescent, luminescent, orisotopic labels. A detailed description of binding affinities andkinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), which focuses onantibody-immunogen interactions.

II. Antibodies which Bind to Same Epitope as or Cross-Compete withAnti-LAP Antibodies

Anti-LAP antibodies which bind to the same or similar epitopes to theantibodies disclosed herein (and thus also cross-compete with theantibodies disclosed herein) may be raised using immunization protocols.The resulting antibodies can be screened for high affinity binding tohuman LAP-TGFβ1. Selected antibodies can then be studied, e.g., in yeastdisplay assay in which sequence variants of LAP-TGFβ1 are presented onthe surface of yeast cells, or by hydrogen-deuterium exchangeexperiments, to determine the precise epitope bound by the antibody.

Antibodies which bind to the same epitope as the anti-LAP antibodiesdescribed herein can also be generated using chimeric constructs, e.g.,chicken-human chimeras of LAP-TGFβ1. Since human and chicken sequencescan be combined to yield a LAP-TGFβ1 protein that folds correctly (asdescribed in Example 2), the method can be used to generate immunogensto specific epitopes of interest on LAP-TGFβ1. With this strategy, themajority of the sequence would be taken from chicken LAP-TGFβ1, withsmall sections of human LAP-TGFβ1 inserted in regions containing thedesired epitope. Exemplary epitopes on LAP-TGFβ1 that can be targetedusing this strategy include, for example, the lower arm of LAP-TGFβ1,the latency loop of LAP-TGFβ1, or an epitope comprising amino acids82-130 of human LAP-TGFβ1. Exemplary chicken-human chimera constructsare described in Example 3. This chimeric protein could be used toimmunize chickens to yield monoclonal antibodies. Since the chickenLAP-TGFβ1 would be recognized as self, the immune response will befocused on the human sequence. Antibodies generated using this approachcan be tested for various functions/properties (e.g., binding toLAP-TGFβ1, inhibiting TGFβ1 activation, binding to ECM, binding to cellssuch as immunosuppressive cells) using standard methods known in theart, e.g., the methods described herein.

The epitope to which an antibody binds can be determined usingart-recognized methods. An anti-LAP antibody is considered to bind tothe same epitope as a reference anti-LAP antibody if it, e.g., contactsone or more of the same residues on human LAP-TGFβ1 as the referenceantibody; contacts one or more of the same residues within at least oneregion of human LAP-TGFβ1 as the reference antibody; contacts a majorityof residues within at least one region of human LAP-TGFβ1 as thereference antibody; contacts a majority of the same residues within eachregion of human LAP-TGFβ1 as the reference antibody; contacts a majorityof the same residues along the entire length of human LAP-TGFβ1 as thereference antibody; contacts all of the same distinct regions of humanLAP-TGFβ1 as the reference antibody; contacts all of the same residuesat any one region on human LAP-TGFβ1 as the reference antibody; orcontacts all of the same residues at all of the same regions of humanLAP-TGFβ1 as the reference antibody.

Techniques for determining antibodies that bind to the “same epitope onhuman LAP-TGFβ1” with the anti-LAP antibodies described herein includex-ray analyses of crystals of antigen:antibody complexes, which providesatomic resolution of the epitope. Other methods monitor the binding ofthe antibody to antigen fragments or mutated variations of the antigenwhere loss of binding due to an amino acid modification within theantigen sequence indicates the epitope component. Methods may also relyon the ability of an antibody of interest to affinity isolate specificshort peptides (either in native three-dimensional form or in denaturedform) from combinatorial phage display peptide libraries or from aprotease digest of the target protein. The peptides are then regarded asleads for the definition of the epitope corresponding to the antibodyused to screen the peptide library. For epitope mapping, computationalalgorithms have also been developed that have been shown to mapconformational discontinuous epitopes.

The epitope or region comprising the epitope can also be identified byscreening for binding to a series of overlapping peptides spanning humanLAP-TGFβ1. Alternatively, the method of Jespers et al. (1994)Biotechnology 12:899 may be used to guide the selection of antibodieshaving the same epitope and therefore similar properties to the anti-LAPantibodies described herein. Using phage display, first, the heavy chainof the anti-LAP antibody is paired with a repertoire of (e.g., human)light chains to select a LAP-binding antibody, and then the new lightchain is paired with a repertoire of (e.g., human) heavy chains toselect a (e.g., human) LAP-binding antibody having the same epitope orepitope region as an anti-LAP antibody described herein. Alternatively,variants of an antibody described herein can be obtained by mutagenesisof cDNA sequences encoding the heavy and light chains of the antibody.

Alanine scanning mutagenesis, as described by Cunningham & Wells (1989)Science 244: 1081, or some other form of point mutagenesis of amino acidresidues in LAP-TGFβ1 may also be used to determine the functionalepitope for an anti-LAP antibody.

The epitope or epitope region (an “epitope region” is a regioncomprising the epitope or overlapping with the epitope) bound by aspecific antibody may also be determined by assessing binding of theantibody to peptides comprising LAP-TGFβ1 fragments. A series ofoverlapping peptides encompassing the LAP-TGFβ1 sequence may besynthesized and screened for binding, e.g. in a direct ELISA, acompetitive ELISA (where the peptide is assessed for its ability toprevent binding of an antibody to LAP-TGFβ1 bound to a well of amicrotiter plate), or on a chip. Such peptide screening methods may notbe capable of detecting some discontinuous functional epitopes.

An epitope may also be identified by MS-based protein footprinting, suchas HDX-MS and Fast Photochemical Oxidation of Proteins (FPOP),structural methods such as X-ray crystal structure determination,molecular modeling, and nuclear magnetic resonance spectroscopy.

Single particle cryo electron microscopy (SP-Cryo-EM) can also be usedto identify the epitope to which an antibody binds. SP-Cryo-EM is atechnique for macromolecular structure analysis which uses a highintensity electron beam to image biological specimens in their nativeenvironment at cryogenic temperature. In recent years, SP-cryo-EM hasemerged as a complementary technique to crystallography and NMR fordetermining near-atomic level structures suitable for application indrug discovery (Renaud et al. Nat Rev Drug Discov 2018; 17:471-92;Scapin et al. Cell Chem Biol 2018; 25:1318-25; Ceska et al. BiochemicalSociety Transactions 2019: p. BST20180267). In addition to highresolution information, SP-Cryo-EM has the further advantage of allowingaccess to larger and more complex biological systems, with thepossibility of characterizing multiple conformational or compositionalsolution states from the same sample, providing insights into morebiologically relevant states of the macromolecule. For imaging, a smallvolume of sample (e.g., 3 μl aliquot) is applied onto a grid andflash-frozen in a liquid ethane bath. The frozen grid is then loadedinto the microscope and hundreds to thousands of images of differentareas of the grids are collected. These images contain two-dimensionalprojections of the biological macromolecule (particles): usingmathematical tools and GPU powered algorithms, the particles areidentified, extracted, and classified; in the subsequent step, thedifferent classes are used to compute one or more 3D reconstructions,corresponding to different conformations, oligomerization or bindingstates if they coexist in the same sample. The individualreconstructions can then be refined to high resolution.

III. Nucleic Acid Molecules

Also provided herein are nucleic acid molecules that encode the anti-LAPantibodies or antigen binding fragments described herein. The nucleicacids may be present in whole cells, in a cell lysate, or in a partiallypurified or substantially pure form. A nucleic acid described herein canbe, for example, DNA or RNA and may or may not contain intronicsequences. In certain embodiments, the nucleic acid is a cDNA molecule.The nucleic acids described herein can be obtained using standardmolecular biology techniques. For antibodies expressed by hybridomas(e.g., hybridomas prepared from transgenic mice carrying humanimmunoglobulin genes as described further below), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), nucleic acid encoding the antibody can berecovered from the library.

In some embodiments, provided herein are nucleic acid molecules thatencode the VH and/or VL sequences, or heavy and/or light chainsequences, of any of the anti-LAP antibodies or antigen bindingfragments described herein. Host cells comprising the nucleotidesequences (e.g., nucleic acid molecules) described herein areencompassed herein. Once DNA fragments encoding VH and VL segments areobtained, these DNA fragments can be further manipulated by standardrecombinant DNA techniques, for example to convert the variable regiongenes to full-length antibody chain genes, to Fab fragment genes or to ascFv gene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (hinge,CH1, CH2 and/or CH3). The sequences of human heavy chain constant regiongenes are known in the art (see e.g., Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

Also provided herein are nucleic acid molecules with conservativesubstitutions that do not alter the resulting amino acid sequence upontranslation of the nucleic acid molecule.

IV. Methods of Production

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof.

Various methods for making monoclonal antibodies described herein areavailable in the art. For example, the monoclonal antibodies can be madeusing the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or any later developments thereof, or by recombinant DNAmethods (U.S. Pat. No. 4,816,567). For example, monoclonal antibodiescan be produced using hybridoma techniques including those known in theart and taught, for example, in Harlow et al, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988);Hammer-ling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties). Methods for producing and screening forspecific antibodies using hybridoma technology are routine and wellknown in the art. In another example, antibodies useful in the methodsand compositions described herein can also be generated using variousphage display methods known in the art, such as isolation from antibodyphage libraries generated using the techniques described in McCaffertyet al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628(1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (e.g., nM range) human antibodies by chain shuffling (Marks etal., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

Human antibodies can be made by a variety of methods known in the art,including phage display methods described above using antibody librariesderived from human immunoglobulin sequences. See also, U.S. Pat. Nos.4,444,887 and 4,716,111; and PCT publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741, the contents of which are herein incorporated by reference intheir entireties. Human antibodies can also be produced using transgenicmice which express human immunoglobulin genes, and upon immunization arecapable of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For an overview of thistechnology for producing human antibodies, see, Lonberg and Huszar,1995, Int. Rev. Immunol. 13:65-93. Phage display technology (McCaffertyet al, Nature 348:552-553 (1990)) also can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. Human antibodiescan also be generated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275, the contents of which are herein incorporatedby reference in their entireties). Completely human antibodies whichrecognize a selected epitope can be generated using a technique referredto as “guided selection.” In this approach a selected non-humanmonoclonal antibody, e.g., a mouse antibody, is used to guide theselection of a completely human antibody recognizing the same epitope(Jespers et al., 1994, Bio/technology 12:899-903).

Chimeric antibodies can be prepared based on the sequence of a murinemonoclonal antibody. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.)

Humanized forms of anti-LAP antibodies (e.g., humanized forms of mouseanti-LAP antibodies) are chimeric antibodies which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodies aretypically human immunoglobulins (recipient antibody) in which residuesfrom a CDR or hypervariable region of the recipient are replaced byresidues from a CDR or hypervariable region of a non-human species(donor antibody) such as mouse, rat, rabbit or nonhuman primate havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Humanized antibodies can compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework can be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond exactly toeither the donor antibody or the consensus framework. As used herein,the term “consensus framework” refers to the framework region in theconsensus immunoglobulin sequence. As used herein, the term “consensusimmunoglobulin sequence” refers to the sequence formed from the mostfrequently occurring amino acids (or nucleotides) in a family of relatedimmunoglobulin sequences (see e.g., Winnaker, From Genes to Clones(Veriagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.Where two amino acids occur equally frequently, either can be includedin the consensus sequence. As used herein, “Vernier zone” refers to asubset of framework residues that may adjust CDR structure and fine-tunethe fit to antigen as described by Foote and Winter (1992, J. Mol. Biol.224:487-499, which is incorporated herein by reference). Vernier zoneresidues form a layer underlying the CDRs and can impact on thestructure of CDRs and the affinity of the antibody. Human immunoglobulin(Ig) sequences that can be used as a recipient are well known in theart.

Framework residues in the human framework regions can be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, including, but not limited to, those described in Jones et al.,Nature 321:522 (1986); Verhoeyen et al., Science 239: 1534 (1988), Simset al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol.196:901 (1987), Carter et al, Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, MolecularImmunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication number WO 91/09967, PCT/: US98/16280, US96/18978,US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596,EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514,5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352,6,204,023, 6,180,370, 5,693,762, 5,530, 101, 5,585,089, 5,225,539;4,816,567, each entirely incorporated herein by reference.

The anti-LAP antibodies generated using the methods described above canbe tested for desired functions, such as particular bindingspecificities, binding affinities, targeted cell populations, usingmethods known in the art and described in the Examples, for example,art-recognized protein-protein binding assays, biochemical screeningassays, immunoassays, and cell-based assays. An aspect of the inventionprovides molecules that may be used to screen for an antibody or antigenbinding fragment that binds LAP, a complex comprising LAP, and/or acomplex comprising LAP-TGFβ1. For example, the molecules in Table 4(i.e., molecules having the amino acid sequence of any of SEQ ID NO: 1,and 198-210) are used to screen or determine binding of at least onebinding protein. In various embodiments, the at least one molecule inTable 4 (i.e., a molecule having the amino acid sequence of any of SEQID NO: 1, and 198-210) and Table 6 (i.e., a molecule having the aminoacid sequence of any of SEQ ID NOs: 211-213) are used to screen ordetermine binding of at least one antibody or antigen binding fragment.

Exemplary assays include, but are not limited to, immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA), FACS,enzyme-linked immunoabsorbent assay (ELISA), bio-layer interferometry(e.g., ForteBio assay), and Scatchard analysis.

Antibody Engineering

Further included are embodiments in which the anti-LAP antibodies orantigen-binding fragments thereof are engineered antibodies to includemodifications to framework residues within the variable domains of theparental monoclonal antibody, e.g., to improve the properties of theantibody or fragment. Typically, such framework modifications are madeto decrease the immunogenicity of the antibody or fragment. This isusually accomplished by replacing non-CDR residues in the variabledomains (i.e., framework residues) in a parental (e.g., rodent) antibodyor fragment with analogous residues from the immune repertoire of thespecies in which the antibody is to be used, e.g., human residues in thecase of human therapeutics. Such an antibody or fragment is referred toas a “humanized” antibody or fragment. In some cases it is desirable toincrease the affinity, or alter the specificity of an engineered (e.g.,humanized) antibody. One approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody or fragment that has undergone somaticmutation can contain framework residues that differ from the germlinesequence from which the antibody is derived. Such residues can beidentified by comparing the antibody or fragment framework sequences tothe germline sequences from which the antibody or fragment is derived.Another approach is to revert to the original parental (e.g., rodent)residue at one or more positions of the engineered (e.g. humanized)antibody, e.g. to restore binding affinity that may have been lost inthe process of replacing the framework residues. (See, e.g., U.S. Pat.Nos. 5,693,762, 5,585,089 and 5,530,101.)

In certain embodiments, the anti-LAP antibodies and antigen-bindingfragments thereof are engineered (e.g., humanized) to includemodifications in the framework and/or CDRs to improve their properties.Such engineered changes can be based on molecular modeling. A molecularmodel for the variable region for the parental (non-human) antibodysequence can be constructed to understand the structural features of theantibody and used to identify potential regions on the antibody that caninteract with the antigen. Conventional CDRs are based on alignment ofimmunoglobulin sequences and identifying variable regions. Kabat et al,(1991) Sequences of Proteins of Immunological Interest, Kabat, et al.;National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIH Publ. No.91-3242; Kabat (1978)Adv. Prot. Chem. 32:1-75; Kabat, et al, (1977) J.Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examinedconformations of the loops in crystal structures of antibodies andproposed hypervariable loops. Chothia, et al, (1987) J Mol. Biol.196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There arevariations between regions classified as “CDRs” and “hypervariableloops”. Later studies (Raghunathan et al., (2012) J. Mol Recog. 25, 3,103-113) analyzed several antibody-antigen crystal complexes andobserved that the antigen binding regions in antibodies do notnecessarily conform strictly to the “CDR” residues or “hypervariable”loops. The molecular model for the variable region of the non-humanantibody can be used to guide the selection of regions that canpotentially bind to the antigen. In practice, the potential antigenbinding regions based on model differ from the conventional “CDR”s or“hyper variable” loops. Commercial scientific software such as MOE(Chemical Computing Group) can be used for molecular modeling. Humanframeworks can be selected based on best matches with the non-humansequence both in the frameworks and in the CDRs. For FR4 (framework 4)in VH, VJ regions for the human germlines are compared with thecorresponding non-human region. In the case of FR4 (framework 4) in VL,J-kappa and J-Lambda regions of human germline sequences are comparedwith the corresponding non-human region. Once suitable human frameworksare identified, the CDRs are grafted into the selected human frameworks.In some cases certain residues in the VL-VH interface can be retained asin the non-human (parental) sequence. Molecular models can also be usedfor identifying residues that can potentially alter the CDRconformations and hence binding to antigen. In some cases, theseresidues are retained as in the non-human (parental) sequence. Molecularmodels can also be used to identify solvent exposed amino acids that canresult in unwanted effects such as glycosylation, deamidation andoxidation. Developability filters can be introduced early on in thedesign stage to eliminate/minimize these potential problems.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. Pat. No.7,125,689.

In particular embodiments, it will be desirable to change certain aminoacids containing exposed side-chains to another amino acid residue inorder to provide for greater chemical stability of the final antibody,so as to avoid deamidation or isomerization. The deamidation ofasparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences andresult in the creation of an isoaspartic acid residue that introduces akink into the polypeptide chain and decreases its stability (isoasparticacid effect). Isomerization can occur at DG, DS, DA or DT sequences. Incertain embodiments, the antibodies of the present disclosure do notcontain deamidation or asparagine isomerism sites. For example, anasparagine (Asn) residue may be changed to Gin or Ala to reduce thepotential for formation of isoaspartate at any Asn-Gly sequences,particularly within a CDR.

A similar problem may occur at a Asp-Gly sequence. Reissner and Aswad(2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation maydebilitate or completely abrogate binding of an antibody to its targetantigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734.

In various embodiment, the asparagine is changed to glutamine (Gin). Itmay also be desirable to alter an amino acid adjacent to an asparagine(Asn) or glutamine (Gin) residue to reduce the likelihood ofdeamidation, which occurs at greater rates when small amino acids occuradjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J.Chromatog. 662:261. In addition, any methionine residues (typicallysolvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe orother amino acids in order to reduce the possibility that the methioninesulfur would oxidize, which could reduce antigen-binding affinity andalso contribute to molecular heterogeneity in the final antibodypreparation. Id. Additionally, in order to prevent or minimize potentialscissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Procombinations found in a CDR to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodieswith such substitutions are subsequently screened to ensure that thesubstitutions do not decrease the affinity or specificity of theantibody for LAP, or other desired biological activity to unacceptablelevels. See Table 1A for exemplary stabilizing CDR variants.

TABLE 1A Exemplary stabilizing CDR variants CDR Residue StabilizingVariant Sequence Asn-Gly Gln-Gly, Ala-Gly, or Asn-Ala (N-G) (Q-G),(A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or Asp-Ala (D-G) (E-G), (A-G),or (D-A) Met Lys, Leu, Ala, or Phe (M) (K), (L), (A), or (F) Asn Gln orAla (N) (Q) or (A) Asn-Pro Gln-Pro, Ala-Pro, or Asn-Ala (N-P) (Q-P),(A-P), or (N-A)

Antibody Engineering of the Fc Region

The antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof disclosed herein (e.g., antibody 20E6 and humanizedversions thereof and antibody and 28G11 and humanized versions thereof)can also be engineered to include modifications within the Fc region,typically to alter one or more properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding, and/or effectorfunction (e.g., antigen-dependent cellular cytotoxicity). Furthermore,the antibodies and antigen-binding fragments thereof disclosed herein(e.g., antibody 20E6 and humanized versions thereof) can be chemicallymodified (e.g., one or more chemical moieties can be attached to theantibody) or be modified to alter its glycosylation, again to alter oneor more properties of the antibody or fragment. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., antibody 20E6 and humanized versions thereof) also includeantibodies and fragments with modified (or blocked) Fc regions toprovide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821;and PCT Publication numbers WO2003/086310; WO2005/120571;WO2006/0057702. Such modifications can be used to enhance or suppressvarious reactions of the immune system, with possible beneficial effectsin diagnosis and therapy. Alterations of the Fc region include aminoacid changes (substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc regions. Changes to the Fc canalso alter the half-life of antibodies in therapeutic antibodies,enabling less frequent dosing and thus increased convenience anddecreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.

In one embodiment, the antibody or antigen-binding fragment of theinvention (e.g., antibody 20E6 and humanized versions thereof) is anIgG4 isotype antibody or fragment comprising a Serine to Prolinemutation at a position corresponding to position 228 (S228P; EU index)in the hinge region of the heavy chain constant region. This mutationhas been reported to abolish the heterogeneity of inter-heavy chaindisulfide bridges in the hinge region (Angal et al. supra; position 241is based on the Kabat numbering system).

In one embodiment of the invention, the hinge region of CH1 is modifiedsuch that the number of cysteine residues in the hinge region isincreased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered, for example, to facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody orantigen-binding fragment of the invention (e.g., antibody 20E6 andhumanized versions thereof and antibody 22F9 and humanized versionsthereof) is mutated to decrease the biological half-life of the antibodyor fragment. More specifically, one or more amino acid mutations areintroduced into the CH2-CH3 domain interface region of the Fc-hingefragment such that the antibody or fragment has impaired Staphylococcylprotein A (SpA) binding relative to native Fc-hinge domain SpA binding.This approach is described in further detail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody or antigen-binding fragment of theinvention (e.g., antibody 20E6 and humanized versions thereof andantibody 20E6 and humanized versions thereof) is modified to increaseits biological half-life. Various approaches are possible. For example,one or more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half-life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody or antigen-binding fragment. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322 can be replaced with a differentamino acid residue such that the antibody has an altered affinity for aneffector ligand and retains the antigen-binding ability of the parentantibody. The effector ligand to which affinity is altered can be, forexample, an Fc receptor or the C1 component of complement. This approachis described in further detail in U.S. Pat. Nos. 5,624,821 and5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication number WO 94/29351.

In yet another example, the Fc region is modified to decrease theability of the antibody or antigen-binding fragment of the invention(e.g., antibody 20E6 and humanized versions thereof and antibody 20E6and humanized versions thereof) to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to decrease the affinity of the antibody orfragment for an Fey receptor by modifying one or more amino acids at thefollowing positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258,264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315,320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340,360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435,437, 438 or 439. This approach is described further in PCT Publicationnumber WO 00/42072. Moreover, the binding sites on human IgG1 for FcγR1,FcγRII, FcγRIII and FcRn have been mapped and variants with improvedbinding have been described (see Shields et al. (2001) J. Biol. Chem.276:6591-6604).

In one embodiment of the invention, the Fc region is modified todecrease the ability of the antibody of the invention (e.g., antibody20E6 and humanized versions thereof) to mediate effector function and/orto increase anti-inflammatory properties by modifying residues 243 and264. In one embodiment, the Fc region of the antibody or fragment ismodified by changing the residues at positions 243 and 264 to alanine.In one embodiment, the Fc region is modified to decrease the ability ofthe antibody or fragment to mediate effector function and/or to increaseanti-inflammatory properties by modifying residues 243, 264, 267 and328.

Altered Effector Function

In some embodiments, the Fc region of an anti-LAP antibody is modifiedto increase or reduce the ability of the antibody or antigen-bindingfragment to mediate effector function and/or to increase/decrease theirbinding to the Fcgamma receptors (FcγRs).

The interaction between the constant region of an antigen bindingprotein and various Fc receptors (FcR) including FcgammaRI (CD64),FcgammaRII (CD32) and FcgammaRIII (CD16) is believed to mediate theeffector functions, such as ADCC and CDC, of the antigen bindingprotein. The Fc receptor is also important for antibody cross-linking,which can be important for anti-tumor immunity.

Effector function can be measured in a number of ways including forexample via binding of the FcgammaRIII to Natural Killer cells or viaFcgammaRI to monocytes/macrophages to measure for ADCC effectorfunction. For example, an antigen binding protein of the presentinvention can be assessed for ADCC effector function in a Natural Killercell assay. Examples of such assays can be found in Shields et al., 2001J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al., 1993 J. Biol.Chem., Vol 268, p 25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.

Human IgG1 constant regions containing specific mutations or alteredglycosylation on residue Asn297 have been shown to reduce binding to Fcreceptors. In other cases, mutations have also been shown to enhanceADCC and CDC (Lazar et al. PNAS 2006, 103; 4005-4010; Shields et al. JBiol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44;1815-1817).

In one embodiment of the present invention, such mutations are in one ormore of positions selected from 239, 332 and 330 (IgG1), or theequivalent positions in other IgG isotypes. Examples of suitablemutations are S239D and I332E and A330L. In one embodiment, the antigenbinding protein of the invention herein described is mutated atpositions 239 and 332, for example S239D and I332E or in a furtherembodiment it is mutated at three or more positions selected from 239and 332 and 330, for example S239D and I332E and A330L. (EU indexnumbering).

In an alternative embodiment of the present invention, there is providedan antibody comprising a heavy chain constant region with an alteredglycosylation profile such that the antigen binding protein has enhancedeffector function. For example, wherein the antibody has enhanced ADCCor enhanced CDC or wherein it has both enhanced ADCC and CDC effectorfunction. Examples of suitable methodologies to produce antigen bindingproteins with an altered glycosylation profile are described in PCTPublication numbers WO2003011878 and WO2006014679 and European patentnumber EP1229125.

In a further aspect, the present invention provides “non-fucosylated” or“afucosylated” antibodies. Non-fucosylated antibodies harbor atri-mannosyl core structure of complex-type N-glycans of Fc withoutfucose residue. These glycoengineered antibodies that lack core fucoseresidue from the Fc N-glycans may exhibit stronger ADCC than fucosylatedequivalents due to enhancement of FcgammaRIIIa binding capacity.

The present invention also provides a method for the production of anantibody according to the invention comprising the steps of: a)culturing a recombinant host cell comprising an expression vectorcomprising the isolated nucleic acid as described herein, wherein therecombinant host cell does not comprise an alpha-1,6-fucosyltransferase;and b) recovering the antigen binding protein. The recombinant host cellmay not normally contain a gene encoding an alpha-1,6-fucosyltransferase(for example yeast host cells such as Pichia sp.) or may have beengenetically modified to inactivate an alpha-1,6-fucosyltransferase.Recombinant host cells which have been genetically modified toinactivate the FUT8 gene encoding an alpha-1,6-fucosyltransferase areavailable. See, e.g., the POTELLIGENT™ technology system available fromBioWa, Inc. (Princeton, N.J.) in which CHOK1SV cells lacking afunctional copy of the FUT8 gene produce monoclonal antibodies havingenhanced antibody dependent cell mediated cytotoxicity (ADCC) activitythat is increased relative to an identical monoclonal antibody producedin a cell with a functional FUT8 gene. Aspects of the POTELLIGENT™technology system are described in U.S. Pat. Nos. 7,214,775 and6,946,292, and PCT Publication numbers WO0061739 and WO0231240. Those ofordinary skill in the art will also recognize other appropriate systems.

It will be apparent to those skilled in the art that such modificationsmay not only be used alone but may be used in combination with eachother in order to further enhance or decrease effector function.

Production of Antibodies with Modified Glycosylation

In still another embodiment, the antibodies or antigen-binding fragmentsof the invention (e.g., antibody 20E6 and humanized versions thereof)comprise a particular glycosylation pattern. For example, anafucosylated or an aglycosylated antibody or fragment can be made (i.e.,the antibody lacks fucose or glycosylation, respectively). Theglycosylation pattern of an antibody or fragment may be altered to, forexample, increase the affinity or avidity of the antibody or fragmentfor a LAP antigen. Such modifications can be accomplished by, forexample, altering one or more of the glycosylation sites within theantibody or fragment sequence. For example, one or more amino acidsubstitutions can be made that result in removal of one or more of thevariable region framework glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity or avidity of the antibody or fragment for antigen. See, e.g.,U.S. Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein (e.g.,antibody 20E6 and humanized versions thereof and antibody 28G11 andhumanized versions thereof) may further include those produced in lowereukaryote host cells, in particular fungal host cells such as yeast andfilamentous fungi have been genetically engineered to produceglycoproteins that have mammalian- or human-like glycosylation patterns(See for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100:5022-5027; Hamilton el al., (2003) Science 301: 1244-1246; Hamilton etal, (2006) Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52(2011); Hamilton et al, Curr Opin Biotechnol. October; 18(5):387-92(2007)). A particular advantage of these genetically modified host cellsover currently used mammalian cell lines is the ability to control theglycosylation profile of glycoproteins that are produced in the cellssuch that compositions of glycoproteins can be produced wherein aparticular N-glycan structure predominates (see, e.g., U.S. Pat. Nos.7,029,872 and 7,449,308). These genetically modified host cells havebeen used to produce antibodies that have predominantly particularN-glycan structures (See for example, Li et al, (2006) Nat. Biotechnol.24: 210-215).

In particular embodiments, the antibodies and antigen-binding fragmentsthereof disclosed herein (e.g., antibody 20E6 and humanized versionsthereof) further include those produced in lower eukaryotic host cellsand which comprise fucosylated and non-fucosylated hybrid and complexN-glycans, including bisected and multiantennary species, including butnot limited to N-glycans such as GlcNAc₍₁₋₄₎Man₃GlcNAc₂;Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂; NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., antibody 20E6 and humanized versionsthereof) may comprise antibodies or fragments having at least one hybridN-glycan selected from the group consisting of GlcNAcMan₅GlcNAc₂;GalGlcNAcMan₅GlcNAc₂; and NANAGalGlcNAcMan₅GlcNAc₂. In particularaspects, the hybrid N-glycan is the predominant N-glycan species in thecomposition.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., antibody 20E6 and humanized versionsthereof and antibody 28G11 and humanized versions thereof) compriseantibodies and fragments having at least one complex N-glycan selectedfrom the group consisting of GlcNAcMan₃GlcNAc₂; GalGlcNAcMan₃GlcNAc₂;NANAGalGlcNAcMan₃GlcNAc₂; GlcNAc₂Man₃GlcNAc₂; GalGlcNAc₂Man₃GlcNAc₂;Gal₂GlcNAc₂Man₃GlcNAc₂; NANAGal₂GlcNAc₂Man₃GlcNAc₂; andNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In particular aspects, the complex N-glycanare the predominant N-glycan species in the composition. In furtheraspects, the complex N-glycan is a particular N-glycan species thatcomprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%,or 100% of the complex N-glycans in the composition. In one embodiment,the antibody and antigen binding fragments thereof provided hereincomprise complex N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%,95%, 97%, 98%, 99%, or 100% of the complex N-glycans comprise thestructure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂, wherein such structure isafucosylated. Such structures can be produced, e.g., in engineeredPichia pastoris host cells.

In particular embodiments, the N-glycan is fucosylated. In general, thefucose is in an α1,3-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,6-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,2-linkage with the Gal at the non-reducing end of theN-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of theN-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end ofthe N-glycan. Therefore, in particular aspects of the above theglycoprotein compositions, the glycoform is in an α1,3-linkage orα1,6-linkage fucose to produce a glycoform selected from the groupconsisting of Man₅GlcNAc₂(Fuc), GlcNAcMan₅GlcNAc₂(Fuc),Man₃GlcNAc₂(Fuc), GlcNAcMan₃GlcNAc₂(Fuc), GlcNAc₂Man₃GlcNAc₂(Fuc),GalGlcNAc₂Man₃GlcNAc₂(Fuc), Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc),NANAGal₂GlcNAc₂Man₃GlcNAc₂(Fuc), and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc);in an α1,3-linkage or α1,4-linkage fucose to produce a glycoformselected from the group consisting of GlcNAc(Fuc)Man₅GlcNAc₂,GlcNAc(Fuc)Man₃GlcNAc₂, GlcNAc₂(FUC₁₋₂₎Man₃GlcNAc₂,GalGlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, Gal₂GlcNAc₂(Fuc1-2)Man3GlcNAc2,NANAGal2GlcNAc2(Fuc₁₋₂)Man₃GlcNAc₂, andNANA₂Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂; or in an α 1,2-linkage fucose toproduce a glycoform selected from the group consisting ofGal(Fuc)GlcNAc₂Man₃GlcNAc₂, Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂,NANAGal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂, andNANA₂Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂.

In further aspects, the antibodies (e.g., humanized antibodies) orantigen-binding fragments thereof comprise high mannose N-glycans,including but not limited to, Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂,Man₅GlcNAc₂, Man₄GlcNAc₂, or N-glycans that consist of the Man₃GlcNAc₂N-glycan structure.

In further aspects of the above, the complex N-glycans further includefucosylated and non-fucosylated bisected and multiantennary species.

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, for example,one that is attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues post-translationally in the Golgi apparatus for N-linkedglycoproteins. N-glycans have a common pentasaccharide core ofMan₃GlcNAc₂ (“Man” refers to mannose; “Glc” refers to glucose; and “NAc”refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). Usually,N-glycan structures are presented with the non-reducing end to the leftand the reducing end to the right. The reducing end of the N-glycan isthe end that is attached to the Asn residue comprising the glycosylationsite on the protein. N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., GlcNAc,galactose, fucose and sialic acid) that are added to the Man₃GlcNAc₂(“Man₃”) core structure which is also referred to as the “trimannosecore”, the “pentasaccharide core” or the “paucimannose core”. N-glycansare classified according to their branched constituents (e.g., highmannose, complex or hybrid). A “high mannose” type N-glycan has five ormore mannose residues. A “complex” type N-glycan typically has at leastone GlcNAc attached to the 1,3 mannose arm and at least one GlcNAcattached to the 1,6 mannose arm of a “trimannose” core. ComplexN-glycans may also have galactose (“Gal”) or N-acetylgalactosamine(“GalNAc”) residues that are optionally modified with sialic acid orderivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminicacid and “Ac” refers to acetyl). Complex N-glycans may also haveintrachain substitutions comprising “bisecting” GlcNAc and core fucose(“Fuc”). Complex N-glycans may also have multiple antennae on the“trimannose core,” often referred to as “multiple antennary glycans.” A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core. The various N-glycans are alsoreferred to as “glycoforms”.

With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”,“G2”, “A1”, and “A2” mean the following. “G-2” refers to an N-glycanstructure that can be characterized as Man₃GlcNAc₂; the term “G-1”refers to an N-glycan structure that can be characterized asGlcNAcMan₃GlcNAc₂; the term “G0” refers to an N-glycan structure thatcan be characterized as GlcNAc₂Man₃GlcNAc₂; the term “G1” refers to anN-glycan structure that can be characterized as GalGlcNAc₂Man₃GlcNAc₂;the term “G2” refers to an N-glycan structure that can be characterizedas Gal₂GlcNAc₂Man₃GlcNAc₂; the term “A1” refers to an N-glycan structurethat can be characterized as NANAGal₂GlcNAc₂Man₃GlcNAc₂; and, the term“A2” refers to an N-glycan structure that can be characterized asNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂-Unless otherwise indicated, the terms G-2”,“G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species thatlack fucose attached to the GlcNAc residue at the reducing end of theN-glycan. When the term includes an “F”, the “F” indicates that theN-glycan species contains a fucose residue on the GlcNAc residue at thereducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2Fall indicate that the N-glycan further includes a fucose residueattached to the GlcNAc residue at the reducing end of the N-glycan.Lower eukaryotes such as yeast and filamentous fungi do not normallyproduce N-glycans that produce fucose.

With respect to multi antennary N-glycans, the term “multi antennaryN-glycan” refers to N-glycans that further comprise a GlcNAc residue onthe mannose residue comprising the non-reducing end of the 1,6 arm orthe 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannoseresidues comprising the non-reducing end of the 1,6 arm and the 1,3 armof the N-glycan. Thus, multi antennary N-glycans can be characterized bythe formulas GlcNAc₍₂₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, orNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. The term “1-4” refers to 1, 2,3, or 4 residues. With respect to bisected N-glycans, the term “bisectedN-glycan” refers to N-glycans in which a GlcNAc residue is linked to themannose residue at the reducing end of the N-glycan. A bisected N-glycancan be characterized by the formula GlcNAc₃Man₃GlcNAc₂ wherein eachmannose residue is linked at its non-reducing end to a GlcNAc residue.In contrast, when a multi antennary N-glycan is characterized asGlcNAc₃Man₃GlcNAc₂, the formula indicates that two GlcNAc residues arelinked to the mannose residue at the non-reducing end of one of the twoarms of the N-glycans and one GlcNAc residue is linked to the mannoseresidue at the non-reducing end of the other arm of the N-glycan.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., antibody 20E6 and humanized versions thereof) may further containone or more glycosylation sites in either the light or heavy chainimmunoglobulin variable region. Such glycosylation sites may result inincreased immunogenicity of the antibody or fragment or an alteration ofthe pK of the antibody due to altered antigen-binding (Marshall et al.(1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002)Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura etal. (2000) Mol Immunol 37:697-706). Glycosylation has been known tooccur at motifs containing an N-X-S/T sequence.

Each antibody or antigen-binding fragment (e.g., 20E6 or humanizedversions thereof) will have a unique isoelectric point (pi), whichgenerally falls in the pH range between 6 and 9.5. The pi for an IgG1antibody typically falls within the pH range of 7-9.5 and the pi for anIgG4 antibody typically falls within the pH range of 6-8.

Each antibody or antigen-binding fragment (e.g., 20E6 or humanizedversions thereof) will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).In general, the T_(MI) (the temperature of initial unfolding) may begreater than 60° C., greater than 65° C., or greater than 70° C. Themelting point of an antibody or fragment can be measured usingdifferential scanning calorimetry (Chen et al (2003) Pharm Res20:1952-60; Ghirlando el al (1999) Immunol Lett 68:47-52) or circulardichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). In afurther embodiment, antibodies and antigen-binding fragments thereof(e.g., antibody 20E6 and humanized versions thereof) are selected thatdo not degrade rapidly. Degradation of an antibody or fragment can bemeasured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes D E (1995) Anal Chem 67:3626-32).

In a further embodiment, antibodies (e.g., antibody 20E6 and humanizedversions thereof) and antigen-binding fragments thereof are selectedthat have minimal aggregation effects, which can lead to the triggeringof an unwanted immune response and/or altered or unfavorablepharmacokinetic properties. Generally, antibodies and fragments areacceptable with aggregation of 25% or less, 20% or less, 15% or less,10% or less, or 5% or less. Aggregation can be measured by severaltechniques, including size-exclusion column (SEC), high performanceliquid chromatography (HPLC), and light scattering.

V. Multispecific Antibodies

Multispecific antibodies (e.g., bispecific antibodies) provided hereininclude at least one binding region for a particular epitope onLAP-TGFβ1 (e.g., human LAP-TGFβ1) as described herein, and at least oneother binding region (e.g., a cancer antigen). Multispecific antibodiescan be prepared as full length antibodies or antibody fragments (e.g.F(ab′)2 antibodies).

Methods for making multispecific antibodies are well known in the art(see, e.g., PCT Publication numbers WO 05117973 and WO 06091209). Forexample, production of full length multispecific antibodies can be basedon the co-expression of two paired immunoglobulin heavy chain-lightchains, where the two chains have different specificities. Varioustechniques for making and isolating multispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, multispecific antibodies can be produced using leucine zippers.Another strategy for making multispecific antibody fragments by the useof single-chain Fv (sFv) dimers has also been reported.

Examples of suitable multispecific molecule platforms include, but arenot limited to, Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-oneAntibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), Fcaband mAh² (F-Star), CovX-body (CovX/Pfizer), Dual Variable Domain(DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts₂Ab(MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee), TvAb(Roche), ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART)(MacroGenics), Dual(ScFv)2-Fab (National Research Center for AntibodyMedicine—China), F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab(Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific(Biotecnol), SEED (EMD Serono), mAb² (F-star), Fab-Fv (UCB-Celltech),Bispecific T Cell Engager (BiTE) (Micromet, Tandem Diabody (Tandab)(Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics),Single-chain Diabody (Academic), TCR-like Antibodies (AIT,ReceptorLogics), COMBODY (Epigen Biotech), dual targeting nanobodies(Ablynx), and Fc-engineered IgG1 (Xencor).

In a particular embodiment, the multispecific antibody comprises a firstantibody (or binding portion thereof) which binds to LAP-TGFβ1derivatized or linked to another functional molecule, e.g., anotherpeptide or protein (e.g., another antibody or ligand for a receptor) togenerate a multispecific molecule that binds to LAP-TGFβ1 and a non-LAPtarget molecule. An antibody may be derivatized or linked to more thanone other functional molecule to generate multispecific molecules thatbind to more than two different binding sites and/or target molecules.To create a multispecific molecule, an antibody disclosed herein can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide,receptor, or binding mimetic, such that a multispecific moleculeresults.

Accordingly, multispecific molecules, for example, bispecific antibodiesand bifunctional antibodies, comprising at least one first bindingspecificity for a particular epitope on LAP-TGFβ1 (e.g., humanLAP-TGFβ1) and a second binding specificity for a second target arecontemplated. In some embodiments, the second target is the secondbinding region specifically binds to a tumor-associated antigen.Tumor-associated antigens are well known in the art. Exemplarytumor-associated antigens include, but are not limited to, AFP, ALK,BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydraseIX, caspase-8, CCR5, CD19, CD20, CD30, CDK4, CEA, cyclin-B1, CYP1B1,EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1,FOLR1, GAGE proteins (e.g., GAGE-1, −2), GD2, GD3, GloboH, glypican-3,GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGEproteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin,ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17,NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5,PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho,SART-1, SART-3, Steap-1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2,Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

In some embodiments, the second binding region of the bispecificantibody specifically binds to CD4, CD8, CD45, CD56, CD14, CD16, CD19,CD20, CD25, CD38, CD11b, CD22, CD30, CD39, CD114, CD23, CD73, CD163,CD206, CD203, CD200R, PD-1, PD-L1, PD-L2, CTLA-4, IDO, TIM-3, LAG-3,TIGIT, PVR, PVRL2, B7H3, B7H4, CSF-1R, VISTA, KIR, OX-40, GITR, 4-1BB,CD40, CD40L, CD27/CD70, CD28, ICOS, CD3, CD56, NKG2DA, NKG2DB, NKG2DC,NKG2DD, NKG2DF, NKG2DH, CD94, NKP46, NKP30, CD33, CD73, CD47, LILRB1,CD91, calreticulin, CD122, GARP, LRRC33, LAP2, LAP3, TGFβ1, TGFβ2,TGFβ3, FAP, cadherin 11 and stanniocalcin 1. In some embodiments, thesecond binding region has agonistic properties when binding to a target,e.g., a TNF family member agonist, OX40 ligand, CD137 ligand, CD137agonist, STING agonist, GITR agonist, ICOS agonist, and CD28 agonist.

In some embodiments, the antibody is a trispecific antibody comprising afirst, second, and third binding region, wherein the first bindingregion comprises the binding specificity (e.g., antigen-binding region)of an anti-LAP antibody described herein, and the second and thirdbinding regions bind to two different targets (or different epitopes onthe same target), for example, the targets described above.

In some embodiments, the antibody is a bifunctional antibody comprisingan anti-LAP antibody described herein and a receptor molecule (i.e., areceptor trap construct such as a TGFβ superfamily ligand receptor(e.g., ActRIIB and variants thereof) or VEGFR).

In one embodiment, the multispecific molecules comprise as a bindingspecificity at least one antibody, or an antibody fragment thereof,including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv. Theantibody may also be a light chain or heavy chain dimer, or any minimalfragment thereof such as a Fv or a single chain construct as describedin Ladner et al. U.S. Pat. No. 4,946,778.

The multispecific molecules can be prepared by conjugating theconstituent binding specificities, e.g., the anti-FcR and anti-LAPbinding specificities, using methods known in the art. For example, eachbinding specificity of the multispecific molecule can be generatedseparately and then conjugated to one another. When the bindingspecificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC). Preferred conjugating agents are SATA and sulfo-SMCC, bothavailable from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the multispecific molecule is a mAb×mAb,mAb×Fab, Fab×F(ab′)₂ or ligand x Fab fusion protein. A multispecificmolecule can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Multispecific moleculesmay comprise at least two single chain molecules. Methods for preparingmultispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the multispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or western blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA). The radioactive isotopecan be detected by such means as the use of a αγ-β counter or ascintillation counter or by autoradiography.

VI. Immunoconjugates

Immunoconjugates comprising the anti-LAP antibodies or antigen bindingfragments thereof described herein can be formed by conjugating theantibodies to another therapeutic agent to form, e.g., an antibody-drugconjugate (ADC). Suitable agents include, for example, a cytotoxic agent(e.g., a chemotherapeutic agent), a toxin (e.g. an enzymatically activetoxin of bacterial, fungal, plant or animal origin, or fragmentsthereof), and/or a radioactive isotope (i.e., a radioconjugate).Additional suitable agents include, e.g., antimetabolites, alkylatingagents, DNA minor groove binders, DNA intercalators, DNA crosslinkers,histone deacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In some embodiments, ADCs with the anti-LAP antibodies orantigen binding fragment thereof described herein (e.g., conjugated to acytotoxic agent) that bind to immunosuppressive cells (e.g., regulatoryT cells) can be used to deplete the immunosuppressive cells from, e.g.,the tumor microenvironment.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,neomycin, and the tricothecenes. Additional examples of cytotoxins orcytotoxic agents include, e.g., taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

In the ADC, the antibody and therapeutic agent preferably are conjugatedvia a cleavable linker such as a peptidyl, disulfide, or hydrazonelinker. More preferably, the linker is a peptidyl linker such asVal-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO:214), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit,Ser, or Glu. The ADCs can be prepared as described in U.S. Pat. Nos.7,087,600; 6,989,452; and 7,129,261; PCT Publication numbers WO02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; andWO 08/103693; U.S. Patent Publication numbers 20060024317; 20060004081;and 20060247295; the disclosures of which are incorporated herein byreference.

A variety of radionuclides are available for the production ofradioconjugated anti-LAP antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Immunoconjugates can also be used to modify a given biological response,and the drug moiety is not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be aprotein or polypeptide possessing a desired biological activity (e.g.,lymphokines, tumor necrosis factor, IFNγ, growth factors).

Immunoconjugates can be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody (see, e.g., PCTpublication number WO94/11026).

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

The anti-LAP antibodies or antigen binding fragments described hereinalso are used for diagnostic purposes. Such antibodies or antigenbinding fragments can be conjugated to an appropriate detectable agentto form an immunoconjugate. For diagnostic purposes, appropriate agentsare detectable labels that include radioisotopes, for whole bodyimaging, and radioisotopes, enzymes, fluorescent labels and othersuitable antibody tags for sample testing.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labels,isotopes, chromophores, fluorescent markers, luminescent markers, metallabels (e.g., for CyTOF, imaging mass cytometry), phosphorescent markersand the like, as well as enzyme labels that convert a given substrate toa detectable marker, and polynucleotide tags that are revealed followingamplification such as by polymerase chain reaction. Suitable enzymelabels include horseradish peroxidase, alkaline phosphatase and thelike. For instance, the label can be the enzyme alkaline phosphatase,detected by measuring the presence or formation of chemiluminescencefollowing conversion of 1,2 dioxetane substrates such as adamantylmethoxy phosphoryloxy phenyl dioxetane (AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-Star®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

Preferably, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide-(i.e. amide-),sulfide-, (sterically hindered), disulfide-, hydrazone-, and etherlinkages. These linkages are nearly non-immunogenic and show reasonablestability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol.13 (2009) 235-244; and PCT Publication numbers WO 2009/059278 and WO95/17886).

Depending on the biochemical nature of the moiety and the antibody,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant of between 50 to 500 amino acids,there are standard procedures in text books describing the chemistry forsynthesis of protein conjugates, which can be easily followed by theskilled artisan (see e.g. Hackenberger, C. P. R., and Schwarzer, D.,Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodimentthe reaction of a maleinimido moiety with a cysteine residue within theantibody or the moiety is used. This is an especially suited couplingchemistry in case e.g. a Fab or Fab′-fragment of an antibody is used.Alternatively in one embodiment coupling to the C-terminal end of theantibody or moiety is performed. C-terminal modification of a protein,e.g. of a Fab-fragment can e.g. be performed as described (Sunbul, M.and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., Chem Bio Chem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C—N bonds is used by Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403). Site specificreaction and covalent coupling can also be achieved by the selectivereaction of terminal amino acids with appropriate modifying reagents.The reactivity of an N-terminal cysteine with benzonitrils (see Ren, H.et al, Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling. Native chemical ligation canalso rely on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali,B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering),65-96).

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al, Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In some embodiments, the moiety attached to an anti-LAP antibody orantigen binding fragment is selected from the group consisting of adetectable moiety, binding moiety, a labeling moiety, and a biologicallyactive moiety.

VII. Assays

The anti-LAP antibodies or antigen binding fragments disclosed hereincan be tested for desired properties, e.g., those described herein,using a variety of assays known in the art.

In one embodiment, the antibodies are or antigen binding fragmentstested for specific binding to LAP-TGFβ1 (e.g., human LAP-TGFβ1).Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-LAP antibodies or antigen binding fragmentsinclude standard assays known in the art, for example, Biacore™ surfaceplasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument(Biacore AB, Uppsala, Sweden) or bio-layer interferometry (e.g.,ForteBio assay), as described in the Examples. In some embodiments, theLAP used in the binding assay is complexed with TGFβ1. In someembodiments, the LAP used in the binding assay is not complexed withTGFβ1. In some embodiments, the LAP used in the binding assay iscomplexed with TGFβ1 and GARP or a fragment of GARP or LRRC33 or afragment of LRRC33. In some embodiments the LAP used in the bindingassay is complexed with TGFβ1 and LTBP (e.g., LTBP1, LTBP3, or LTBP4) ora fragment of LTBP.

In one embodiment, the antibodies or antigen binding fragments aretested for the ability to bind to cells that have been transfected withLAP-TGFβ1. In some embodiments the cells have also been transfected withGARP or LRRC33.

In one embodiment, the antibodies or antigen binding fragments arescreened for the ability to bind to the surface of beads that have beencoated with LAP.

In one embodiment, the antibodies or antigen binding fragments arescreened for the ability to bind to LAP on cells expressing a heparinsulfate glycoprotein such as syndecan-4. For example, heparin sulfateglycoprotein-expressing cells are incubated with LAP or with LAPcomplexed to LTBP (e.g., LTBP1, LTBP3, or LTBP4) and the antibodies arescreened for binding by flow cytometry.

In one embodiment, the antibodies or antigen binding fragments aretested for the ability to bind or affect TGFβ1. In one embodiment, theantibodies are screened for the ability to bind or affect TGFβ2. In oneembodiment, the antibodies are tested for the ability to bind or affectTGFβ3.

In another embodiment, the antibodies or antigen binding fragments aretested for their effects on TGFβ activation (e.g., inhibition,stimulation, or no effect). In some embodiments, TGFβ1 activation ismediated by the binding of integrins including, but not limited, toαvβ6, αvβ8, αvβ, or αvβ1. In some embodiments, TGFβ1 activation ismediated by matrix metalloproteases including, but not limited to, MMP2and MMP9. In some embodiments, TGFβ1 activation is mediated bythrombospondin. In some embodiments, TGFβ1 activation is mediated byserum proteases. In some embodiments, TGFβ1 activation is mediated byheat, by shear forces, by a shift in pH or by ionizing radiation. Insome embodiments, TGFβ1 activation is mediated by reactive oxygenspecies (ROS). The source of LAP in the activation assays can be LAP onthe surface of a transfected cell line, LAP on the surface of a cellpopulation that expresses LAP endogenously or in response to specificstimuli, LAP bound to extracellular matrix, LAP in solution (e.g.,recombinant LAP), either complexed with TGFβ1 or without TGFβ1 orcomplexed with TGFβ1 and an anchor protein, such as GARP, LRRC33, LTBP1,LTBP3, or LTBP4. LAP-TGFβ1 can be purchased from R&D Systems or can beisolated from cell supernatants. The effect an antibody has on TGFβ1activation can be determined, for example, using an ELISA (e.g., asdescribed in Example 4) which measures levels of active TGFβ1 underdifferent conditions (e.g., with or without antibody). The effect anantibody has on LAP-TGFβ1 activation can also be determined using areporter cell line that expresses TGFβ receptor and responds to matureTGFβ.

In another embodiment, the antibodies or antigen binding fragments aretested for the ability to bind LAP in the extracellular matrix. Suitablemethods for determining whether antibodies bind to LAP in theextracellular matrix include in vitro assays, wherein cells (e.g., P3U1cells transfected with LAP-TGFβ) are cultured to lay down ECM on cultureplates and subsequently removed, and labeled antibodies are tested fortheir ability to bind to the LAP and ECM left on the culture platesurface (e.g., as described in Example 5). Similar assays can be runusing fibroblast cell lines or other cells that are known to secreteLAP-TGFβ and extracellular matrix components. In some embodiments,whether or not the anti-LAP antibodies bind to or do not bind to ECM canbe determined by an ELISA, where the ECM has been shown to expresslatent TGFβ using commercially available antibodies.

In another embodiment, the antibodies or antigen binding fragments aretested for their ability to bind to particular cell types, e.g., immunecells (e.g., immunosuppressive cells, leukocytes) or platelets. Thebinding of antibodies or antigen binding fragments to certain leukocytepopulations (e.g., Tregs, macrophages, MDSCs, GARP-negative cells) canbe determined using flow cytometry, for example, as described inExamples 7.

Antibodies or antigen binding fragments can also be tested for theirability to inhibit the proliferation or viability of cells (either invivo or in vitro), such as tumor cells, using art-recognized methods(e.g., 3H-thymidine incorporation, immunohistochemistry withproliferation markers, animal cancer models).

Antibodies or antigen binding fragments can also be tested for theiranti-tumor activity in vivo (e.g., as monotherapy or combinationtherapy), using syngeneic tumor models well known in the art, such asthe CT26 colorectal tumor model, EMT6 breast cancer model, and 4T1breast cancer tumor metastasis model. Anti-LAP antibodies can also betested in tumor xenogragft models which are known to be inhibited byanti-TGFβ antibodies (e.g., Detroit 562 tumor xenograft model).Exemplary methods for treating these models with anti-LAP antibodies aredescribed, e.g., in Examples 12-16.

Exemplary criteria for determining whether an anti-LAP antibody orantigen binding fragment exhibits certain properties (e.g., binding,inhibition of activation, activation) are shown in Table IB.

Table IB.

TABLE 1B Antibody Property Positive Binding to cells or ECM, as 2 SDabove the mean of a negative assessed by ELISA control Binding to celltypes, as 2 SD above the mean (MFI on a homoge- assessed by flowcytometry neous cell line or cell population) of a negative controlBinding to TGFβ by a ≥100-fold difference in affinity binding assay(e.g., bio- relative to a negative control layer interferometryInhibition of TGFβ1 activation ≥50% reduction in mature TGFβ1 levels inan in vitro culture relative to negative control when tested at antibodyconcentrations of 8 ug/mL Activation of TGFβ1 ≥2-fold increase in matureTGFβ1 levels in an in vitro culture relative to negative control whentested at antibody concentrations of 8 ug/mL

VIII. Compositions

Also provided herein are compositions (e.g., pharmaceuticalcompositions) comprising the anti-LAP antibodies or antigen bindingfragments described herein, immunoconjugates comprising the same, orbispecific antibodies comprising the same, and a carrier (e.g.,pharmaceutically acceptable carrier). Such compositions are useful forvarious therapeutic applications.

In some embodiments, pharmaceutical compositions disclosed herein caninclude other compounds, drugs, and/or agents used for the treatment ofvarious diseases (e.g., cancer, fibrosis, autoimmune diseases). Suchcompounds, drugs, and/or agents can include, for example, an anti-canceragent, a chemotherapeutic agent, an immunosuppressive agent, animmunostimulatory agent, an immune checkpoint inhibitor, and/or ananti-inflammatory agent. Exemplary compounds, drugs, and agents that canbe formulated together or separately with the anti-LAP antibodies orantigen binding fragments described herein are described in the nextsection (i.e., Section IX; Uses and Methods).

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition described herein may also include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. A pharmaceutical composition maycomprise a preservative or may be devoid of a preservative.Supplementary active compounds can be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody or antigen binding fragment, thedosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5or 10 mg/kg, of the host body weight. An exemplary treatment regimeentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months.

An antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

The therapeutically effective dosage of an anti-LAP antibody or antigenbinding fragment in various embodiments results in a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. In the context of cancer, atherapeutically effective dose preferably results in increased survival,and/or prevention of further deterioration of physical symptomsassociated with cancer. A therapeutically effective dose may prevent ordelay onset of cancer, such as may be desired when early or preliminarysigns of the disease are present.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies describedherein include intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Alternatively, an antibody or antigen binding fragment described hereincan be administered via a non-parenteral route, such as a topical,epidermal or mucosal route of administration, for example, intranasally,orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules for use withanti-LAP antibodies described herein include: U.S. Pat. No. 4,487,603,which discloses an implantable micro-infusion pump for dispensingmedication at a controlled rate; U.S. Pat. No. 4,486,194, whichdiscloses a therapeutic device for administering medicants through theskin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-LAP antibodies or antigen bindingfragments described herein can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds described herein cross the BBB (if desired, e.g.,for brain cancers), they can be formulated, for example, in liposomes.For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos.4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one ormore moieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995)Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

IX. Uses and Methods

The antibodies, antibody compositions, and methods described herein havenumerous in vitro and in vivo utilities.

For example, provided herein is a method of treating cancer comprisingadministering to a subject in need thereof an anti-LAP antibody orantigen binding fragment described herein, such that the subject istreated, e.g., such that growth of cancerous tumors is inhibited orreduced and/or that the tumors regress and/or that prolonged survival isachieved.

In one embodiment, provided herein is a method of treating cancercomprising administering to a subject in need thereof an effectiveamount (e.g., a therapeutically effective amount) of an anti-LAPantibody described herein (or a bispecific antibody or immunoconjugatecomprising the antibody). In some embodiments, the subject isadministered a further therapeutic agent. In some embodiments, thefurther therapeutic agent is selected from the group consisting of: ananti-PD-1 antibody or an antigen binding fragment thereof, an anti-LAG3antibody or an antigen biding portion thereof, an anti-VISTA antibody oran antigen binding fragment thereof, an anti-BTLA antibody or an antigenbinding fragment thereof, an anti-TIM3 antibody or an antigen bindingfragment thereof, an anti-CTLA4 antibody or an antigen binding fragmentthereof, an anti-HVEM antibody or an antigen binding fragment thereof,an anti-CD27 antibody or an antigen binding fragment thereof, ananti-CD137 antibody or an antigen binding fragment thereof, an anti-OX40antibody or an antigen binding fragment thereof, an anti-CD28 antibodyor an antigen binding fragment thereof, an anti-PDL1 antibody or anantigen binding fragment thereof, an anti-PDL2 antibody or an antigenbinding fragment thereof, an anti-GITR antibody or an antigen bindingfragment thereof, an anti-ICOS antibody or an antigen binding fragmentthereof, an anti-SIRPα antibody or an antigen binding fragment thereof,an anti-ILT2 antibody or an antigen binding fragment thereof, ananti-ILT3 antibody or an antigen binding fragment thereof, an anti-ILT4antibody or an antigen binding fragment thereof, an anti-ILT5 antibodyor an antigen binding fragment thereof, and an anti-4-1BB antibody or anantigen binding fragment thereof. In some embodiments, anti-PD 1antibody or antigen binding fragment thereof is pembrolizumab or anantigen biding fragment thereof. The heavy and light chain sequences ofpembrolizumab are set forth in SEQ ID NOs: 240 and 241, respectively. Insome embodiments, the further therapeutic agent is nivolumab. In variousembodiments, the heavy and light chain sequences of nivolumab are setforth in comprising SEQ ID NOs: 246 and 247.

In some embodiments, the cancer is characterized by abnormal TGFβactivity. In some embodiments, the cancer is associated with fibrosis.In some embodiments, the cancer is associated with infiltration of CD4+regulatory T cells. In some embodiments, the cancer is associated withinfiltration of CD8+ regulatory T cells. In some embodiments, the canceris associate with infiltration of regulatory B cells. In someembodiments, the cancer is associated with infiltration ofmyeloid-derived suppressor cells. In some embodiments, the cancer isassociated with infiltration of tumor-associated macrophages. In someembodiments, the cancer is associated with infiltration of innatelymphoid cells. In some embodiments, the cancer is associated withinfiltration of cancer-associated fibroblasts. In some embodiments, thecancer is associated with a radiation-related increase in the above celltypes.

In some embodiments, the cancer is associated with an increased TGFβ1activation signature. In some embodiments the cancer is associated withan EMT or an EMT signature. In some embodiments the cancer is associatedwith a tumor exhibiting an EMT or an EMT signature and immuneinfiltration. In some embodiments the cancer is associated with a tumorprofile of immune exclusion. In some embodiments, the cancer isassociated with increased LAP expression. In some embodiments, thecancer is associated with increased GARP expression. In someembodiments, the cancer is associated with increased LRRC33 expression.

Cancers whose growth may be inhibited using the anti-LAP antibodiesdescribed herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer (e.g. estrogen-receptor positive breast cancer HER2-positivebreast cancer; triple negative breast cancer); cancer of the peritoneum;cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; liver cancer (e.g. hepatocellular carcinoma; hepatoma);intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; lung cancer (e.g., small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; teratocarcinoma; testicular cancer; thyroidcancer; uterine or endometrial cancer; cancer of the urinary system;vulval cancer; as well as other carcinomas and sarcomas; as well asB-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastsleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), tumors of primitiveorigins and Meigs' syndrome.

Additional cancers which can be treated using the anti-LAP antibodies orantigen binding fragments described herein include metastatic pancreaticcancer, metastatic adenocarcinoma of the pancreas, stomach cancer,fibrotic cancer, glioma, malignant glioma, diffuse intrinsic pontineglioma, recurrent childhood brain neoplasm renal cell carcinoma,clear-cell metastatic renal cell carcinoma, metastatic castrationresistant prostate cancer, stage IV prostate cancer, metastaticmelanoma, malignant melanoma, recurrent melanoma of the skin, melanomabrain metastases, malignant melanoma of head and neck, squamous cellnon-small cell lung cancer, metastatic breast cancer, follicularlymphoma, advanced B-cell NHL, HL including diffuse large B-celllymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adultacute myeloid leukemia in remission, adult acute myeloid leukemia withInv(16)(p13. Iq22), CBFB-MYH11, adult acute myeloid leukemia witht(16:16) (p13.1:q22), CBFB-MYH11, adult acute myeloid leukemia witht(8:21)(d22:q22), RUNX1-RUNX1T1, adult acute myeloid leukemia witht(9:11)(p22:q23), MLLT3-MLL, adult acute promyelocytic leukemia withtO15:17)(q22:q12), PML-RARA, alkylating agent-related acute myeloidleukemia, Richter's syndrome, adult glioblastoma, adult gliosarcoma,recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrentewing sarcoma/peripheral primitive neuroectodermal tumor, recurrentneuroblastoma, recurrent osteosarcoma, colorectal cancer, MSI positivecolorectal cancer, MSI negative colorectal cancer, nasopharyngealnonkeratinizing carcinoma, recurrent nasopharyngeal undifferentiatedcarcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma;cervical squamous cell carcinoma, recurrent cervical carcinoma, analcanal squamous cell carcinoma, metastatic anal canal carcinoma,recurrent anal canal carcinoma, recurrent head and neck cancer, squamouscell of head and neck, head and neck squamous cell carcinoma (HNSCC),ovarian carcinoma, colon cancer, advanced GI cancer, gastricadenocarcinoma, gastroesophageal junction adenocarcinoma, boneneoplasms, soft tissue sarcoma, bone sarcoma, thymic carcinoma,urothelial carcinoma, merkel cell carcinoma, recurrent merkel cellcarcinoma, mycosis fungoides, Sezary syndrome, neuroendocrine cancer,nasopharyngeal cancer, basal cell skin cancer, squamous cell skincancer, dermatofibrosarcoma trotuberans, glioma, mesothelioma,myelodysplastic syndromes (MDS), myelofibrosis (MF), myeloproliferativeneoplasms, and acute myeloid leukemia (AML).

Cancers may be metastatic or may be primary cancers. Cancers may bedesmoplastic or non-desmoplastic. Cancers may be recurrent cancers.

In some embodiments, the anti-LAP antibodies or antigen bindingfragments described herein are used to treat myelodysplastic syndromes(MDS). MDS are a diverse group of malignant disorders marked by bonemarrow failure due to defective hematopoiesis and production ofdysplastic cells. TGFβ is a primary driver in MDS (Geyh et al.,Haematologica 2018; 103:1462-71) and agents that inhibit the function ofTGFβ have been proposed as therapeutics (Mies et al., Curr Hematol MaligRep 2016; 11:416-24). Furthermore, MDSCs are known to be dysregulated inMDS (Chen et al., JCI 2013; 123:4595-611) and agents that reduce MDSClevels in the bone marrow are potential therapeutics.

In some embodiments, the anti-LAP antibodies or antigen bindingfragments described herein are used to treat myelofibrosis, which isanother myeloid malignancy in which TGFβ1 plays a central role(Mascarenhas et al., Leukemia & Lymphoma 2014; 55:450-2).

In some embodiments, the cancer is resistant to checkpoint inhibitor(s).In some embodiments, the cancer is intrinsically refractory or resistant(e.g., resistant to a PD-1 pathway inhibitor, PD-1 pathway inhibitor, orCTLA-4 pathway inhibitor). In some embodiments, the resistance orrefractory state of the cancer is acquired. In some embodiments, theanti-LAP antibodies or antigen binding fragments described herein can beused in combination with checkpoint inhibitors to overcome resistance ofthe cancer to the checkpoint inhibitors. In some embodiments, theanti-LAP antibodies or antigen binding fragments described herein can beused to treat tumors with a mesenchymal and/or EMT signature togetherwith checkpoint inhibitors in combination or sequentially with agentsthat induce a mesenchymal phenotype, such as MAPK pathway inhibitors.

In some embodiments, the anti-LAP antibodies or antigen bindingfragments described herein are used to enhance the viability of immunecells ex vivo, e.g., in adoptive NK cell transfer. Accordingly, in someembodiments, anti-LAP antibodies are used in combination with adoptivelytransferred NK cells to treat cancer.

In some embodiments, the anti-LAP antibodies or antigen bindingfragments described herein are used to treat tumors with MHC loss or MHCdown-regulation, as monotherapy or in combination with NK activating orenhancing treatment. In some embodiments, the anti-LAP antibodiesdescribed herein are used to treat checkpoint inhibitor resistant tumorsin combination with NK activating or enhancing treatment.

Also provided herein is a method of treating cancer associated with anincreased number of circulating platelets or an increased platelet tolymphocyte ratio comprising administering to a subject in need thereofan effective amount of an antibody or antigen binding fragment whichspecifically binds to LAP, wherein the antibody binds to platelets butdoes not cause platelet aggregation or platelet degranulation.

The ability of a compound to inhibit cancer can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit using in vitro assaysknown to the skilled practitioner. A therapeutically effective amount ofa therapeutic compound can decrease tumor size, or otherwise amelioratesymptoms in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular composition orroute of administration selected.

Also encompassed are methods for detecting the presence of LAP-TGFβ1 ina sample (e.g., a tumor biopsy sample), or measuring the amount ofLAP-TGFβ1 in sample, comprising contacting the sample (e.g., tumortissue) and a control sample (e.g., corresponding healthy tissue) withan antibody (e.g., monoclonal antibody) or antigen binding fragmentwhich specifically binds to LAP-TGFβ1 under conditions that allow forformation of a complex between the antibody or portion thereof andLAP-TGFβ1. The formation of a complex is then detected, wherein adifference in complex formation between the sample compared to thecontrol sample is indicative of the presence of LAP-TGFβ1 in the sample.The anti-LAP antibodies or antigen binding fragments described hereincan also be used to purify LAP-TGFβ1 via immunoaffinity purification.

Diagnostic applications of the anti-LAP antibodies described herein arealso contemplated.

In one embodiment, provided herein is a method of diagnosing a cancerassociated with regulatory T cell infiltration comprising contacting abiological sample from a patient afflicted with the cancer with ananti-LAP antibody or antigen binding fragment described herein whichbinds to regulatory T cells, wherein positive staining with the antibodyindicates the cancer is associated with regulatory T cell infiltration.

In another embodiment, provided herein is a method of diagnosing acancer associated with GARP-negative suppressive cells comprisingcontacting a biological sample from a patient afflicted with the cancerwith an anti-LAP antibody or antigen binding fragment described hereinwhich binds to GARP-negative suppressive cells, wherein positivestaining with the antibody and negative staining with an anti-GARPantibody indicates the cancer is associated with GARP-negativesuppressive cells.

In another embodiment, provided herein is a method of selecting apatient afflicted with cancer for treatment with an anti-LAP antibody orantigen binding fragment described herein, comprising contacting abiological sample from the patient with the antibody, wherein positivestaining with the antibody indicates the cancer is amenable to treatmentwith the antibody.

In another embodiment, provided herein is a method of determining theresponse of a patient afflicted with cancer to treatment with ananti-LAP antibody or antigen binding fragment described hereincomprising contacting a biological sample from the patient with theantibody, wherein reduced staining with the antibody indicates thecancer is responding to treatment with the antibody.

In another embodiment, provided herein is a method of determiningwhether a cancer in a patient has metastasized comprising (a)identifying a patient having a cancer, (b) administering a labeled(e.g., radiolabeled) anti-LAP antibody or antigen binding fragmentdescribed herein to the patient and determining the biodistribution ofthe labeled anti-LAP antibody, and (c) periodically repeating step (b)to determine whether the biodistribution of the labeled anti-LAPantibody has changed, wherein a change in the biodistribution of thelabeled anti-LAP antibody is indicative that the cancer hasmetastasized.

Also provided are methods of treating fibrosis with the anti-LAPantibodies described herein. In one embodiment, provided herein is amethod of treating fibrosis comprising administering to a subject inneed thereof an effective amount of an antibody or antigen bindingfragment described herein. In some embodiments, the fibrosis isassociated with cancer. In some embodiments, the fibrosis is associatedwith increased levels of myeloid-derived suppressor cells (e.g.Fernandez et al., Eur Respir J 2016; 48:1171-83).

Exemplary fibrotic disorders which can be treated with any of theanti-LAP antibodies or antigen binding fragment described hereininclude, but are not limited to, heart fibrosis, muscle fibrosis, skinfibrosis, liver fibrosis, soft tissue (e.g., mediastinum orretroperitoneum) fibrosis, renal fibrosis, bone marrow fibrosis,intestinal fibrosis, joint (e.g., knee, shoulder or other joints)fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis,retroperitoneal fibrosis, progressive massive fibrosis, pipestemfibrosis, nephrogenic systemic fibrosis, Crohn's disease, keloid, oldmyocardial infarction, scleroderma/systemic sclerosis, subepithelialfibrosis, arthrofibrosis, some forms of adhesive capsulitis,proliferative fibrosis, viral hepatitis induced fibrosis, drug-inducedfibrosis, radiation-induced fibrosis, and fibrosis associated withcancer.

Also provided herein is a method of reducing the number ofimmunosuppressive cells in a patient before, during, or aftertransplantation comprising administering an effective amount of any ofthe anti-LAP antibodies or antigen binding fragments described herein toa patient before undergoing transplantation, during transplantation,and/or after transplantation. In some embodiments, the anti-LAPantibodies or antigen binding fragments improve graft survival.

Inhibition of TGFβ has been shown to restore regenerative failure byreducing senescence and enhancing liver regeneration, in a model ofacute liver disease (acetaminophen injury mouse model) (Bird et al., SciTransl Med 2018; 10:eaan1230). Accordingly, also provided herein is amethod of increasing the regenerative response in acute organ injury(e.g., acute liver injury) comprising administering to a subject withacute organ injury an effective amount of the anti-LAP antibodies orantigen binding fragments described herein.

Aberrant activation of TGFβ has been shown to initiate the onset oftemporomandibular joint osteoarthritis (Zheng et al., Bone Res 2018;6:26). Accordingly, also provided herein is a method of treating apatient with temporomandibular joint osteoarthritis comprisingadministering to the patient an effective amount of the anti-LAPantibodies or antigen binding fragments described herein to treat thetemporomandibular joint osteoarthritis.

LAP-TGFβ1 has also been shown to mediate the differentiation of CD4+effector cells into productively and latently infected central memory Tcells during HIV-1 infection (Cheung et al., J Viol 2018; 92:e01510-17).Accordingly, also provided herein is a method of treating a patient withHIV-1 infection (or a patient at risk of developing HIV-1 infection)comprising administering to the patient an effective amount of theanti-LAP antibodies or antigen binding fragments described herein totreat the HIV-1 infection (e.g., inhibit differentiation of CD4+effector cells into productively and latently infected central memory Tcells).

TGFβ-expressing macrophages and suppressive regulatory T cells have beenshown to be altered in the peritoneal fluid of patients withendometriosis (Hanada et al., Reprod Biol Endocrinol 2018; 16:9),suggesting that targeting LAP-TGFb1 expressed on these cells may bebeneficial for treating the disorder. Accordingly, also provided hereinis a method of treating a patient with endometriosis comprisingadministering to the patient an effective amount of the anti-LAPantibodies or antigen binding fragments described herein to treat theendometriosis.

LAP-TGFβ1-expressing CD4+ T cells and CD14+ monocytes and macrophageshave been shown to be increased in patients carrying multi drugresistant Mycobacterium tuberculosis (Basile et al., Clin Exp Immunol2016; 187:160), suggesting that targeting LAP-TGFβ1 expressed on thesecells may be beneficial for treating the infection. Accordingly, alsoprovided herein is a method of treating a patient with multi drugresistant Mycobacterium tuberculosis comprising administering to thepatient an effective amount of the anti-LAP antibodies or antigenbinding fragments described herein (e.g., anti-LAP antibodies whichinhibit LAP-TGFβ1 activation) to treat the infection.

In some embodiments, the anti-LAP antibodies or antigen bindingfragments described herein are used to treat β-thalassemia, a disorderin which TGFβ superfamily members have been implicated in defectiveerythropoiesis (Dussiot et al. Nat Med 2014; 20:398-407).

In certain embodiments, the anti-LAP antibody or antigen bindingfragment can be used as monotherapy to treat a disease or disorder(e.g., cancer). Alternatively, an anti-LAP antibody or antigen bindingfragment can be used in conjunction with another agent or therapy, e.g.,an anti-cancer agent, a chemotherapeutic agent, an immunosuppressiveagent, an immunostimulatory agent, an immune checkpoint inhibitor, ananti-inflammatory agent, or a cell therapy, as described in more detailbelow.

Combination Therapy

The anti-LAP antibodies or antigen binding fragments described hereincan be used in combination with various treatments or agents (or in thecontext of a multispecific antibody or bifunctional partner) known inthe art for the treatment of cancer, as described below.

Suitable anti-cancer agents for use in combination therapy with theanti-LAP antibodies or antigen binding fragments described hereininclude, but are not limited to, surgery, chemotherapeutic agents,growth inhibitory agents, cytotoxic agents, radiotherapy and agents usedin radiation therapy, anti-angiogenesis agents, apoptotic agents,anti-tubulin agents, and other agents to treat cancer, such asanti-HER-2 antibodies (e.g., HERCEPTIN®), anti-CD20 antibodies, anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA®)),platelet derived growth factor inhibitors (e.g., GLEEVEC (ImatinibMesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe following targets PD 1, PDL1, PDL2 (e.g., pembrolizumab; nivolumab;MK-3475; AMP-224; MPDL3280A; MEDI0680; MSB0010718C; and/or MEDI4736);CTLA4 (e.g., tremelimumab (PFIZER) and ipilimumab); LAG3 (e.g.,BMS-986016); CD 103; TIM-3 and/or other TIM family members; CEACAM-1and/or other CEACAM family members, ErbB2, ErbB3, ErbB4, PDGFR-beta,BlyS, APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, PARP inhibitors(e.g., AZD-2281, Lynparza Olaparib, Rubraca Rucaparib; (Zejula)niraparib), DNA damage repair inhibitors (e.g., ATMi, ATRi, DNAPKi), andother bioactive and organic chemical agents. Combinations thereof arealso specifically contemplated for the methods described herein.

Suitable chemotherapeutic agents for use in combination therapy with theanti-LAP antibodies or antigen binding fragments described hereininclude, but are not limited to, alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; temozolomide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicin omegal1 (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potflromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin,vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also suitable for use in combination with the anti-LAP antibodies orantigen binding fragments described herein are drugs targetingepigenetic regulators, such as HD AC inhibitors, bromodomain inhibitors,and E3 ligase (e.g., cereblon) inhibitors (e.g., lenalidomide,pomalidomide, and thalidomide).

Suitable anti-inflammatory agents for use in combination therapy withthe anti-LAP antibodies or antigen binding fragments described hereininclude, but are not limited to, aspirin and other salicylates, Cox-2inhibitors (e.g., rofecoxib and celecoxib), NSAIDs (such as ibuprofen,fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen,diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin),anti-IL6R antibodies, anti-IL8 antibodies, anti-IL15 antibodies,anti-IL15R antibodies, anti-CD4 antibodies, anti-CD11a antibodies (e.g.,efalizumab), anti-alpha-4/beta-1 integrin (VLA4) antibodies (e.g.,natalizumab), CTLA4-Ig for the treatment of inflammatory diseases,prednisolone, prednisone, disease modifying antirheumatic drugs (DMARDs)such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidinesynthesis inhibitors (e.g., leflunomide), IL-1 receptor blocking agents(e.g., anakinra), TNF-α blocking agents (e.g., etanercept, infliximab,and adalimumab), and the like.

Suitable immunomodulatory agents (e.g., immunostimulatory andimmunosuppressive agents) include, but are not limited to, cyclosporine,azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroidssuch as prednisone, methotrexate, gold salts, sulfasalazine,antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine,6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506),OKT3, anti-thymocyte globulin, thymopentin, thymosin-α, antibodies thatbind to p75 of the IL-2 receptor, antibodies that bind to MHC, CD2, CD3,CD4, CD7, CD28, B7, CD40, CD45, IFN-γ, TNF-α, IL-4, IL-5, IL-6R, IL-6,IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58, or antibodies binding totheir ligands, soluble IL-15R, IL-10, B7 molecules (B7-1, B7-2, variantsthereof, and fragments thereof), ICOS, OX40, an inhibitor of a negativeT cell regulator (such as an antibody against CTLA4), and the like.

Additional immunosuppressive agents include, for example, anti-TNFagents such as etanercept, adalimumab and infliximab, and steroids.Examples of specific natural and synthetic steroids include, forexample: aldosterone, beclomethasone, betamethasone, budesonide,cloprednol, cortisone, cortivazol, deoxycortone, desonide,desoximetasone, dexamethasone, difluorocortolone, fluclorolone,flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl,fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone,fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone,methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol,and triamcinolone.

Suitable immunostimulatory agents for use in combination therapy withthe anti-LAP antibodies or antigen binding fragments described hereininclude, for example, compounds capable of stimulating antigenpresenting cells (APCs), such as dendritic cells (DCs) and macrophages.For example, suitable immunostimulatory agents are capable ofstimulating APCs, so that the maturation process of the APCs isaccelerated, the proliferation of APCs is increased, and/or therecruitment or release of co-stimulatory molecules (e.g., CD80, CD86,ICAM-1, MHC molecules and CCR7) and pro-inflammatory cytokines (e.g.,IL-1β, IL-6, IL-12, IL-15, and IFN-γ) is upregulated. Suitableimmunostimulatory agents are also capable of increasing T cellproliferation. Such immunostimulatory agents include, but are not belimited to, CD40 ligand; FLT 3 ligand; cytokines, such as IFN-α, IFN-β,IFN-γ and IL-2; colony-stimulating factors, such as G-CSF (granulocytecolony-stimulating factor) and GM-CSF (granulocyte-macrophagecolony-stimulating factor); an anti-CTLA-4 antibody, anti-PD 1 antibody,anti-4 IBB antibody, or anti-OX-40 antibody; LPS (endotoxin); ssRNA;dsRNA; Bacille Calmette-Guerin (BCG); Levamisole hydrochloride; andintravenous immune globulins. In one embodiment an immunostimulatoryagent may be a Toll-like Receptor (TLR) agonist. For example theimmunostimulatory agent may be a TLR3 agonist such as double-strandedinosine:cytosine polynucleotide (Poly I:C, for example available asAmpligen™ from Hemispherx Bipharma, PA, US or Poly IC:LC from Oncovir)or Poly A:U; a TLR4 agonist such as monophosphoryl lipid A (MPL) orRC-529 (for example as available from GSK, UK); a TLR5 agonist such asflagellin; a TLR7 or TLR8 agonist such as an imidazoquinoline TLR7 orTLR 8 agonist, for example imiquimod (e.g., Aldara™) or resiquimod andrelated imidazoquinoline agents (e.g., as available from 3MCorporation); or a TLR 9 agonist such as a deoxynucleotide withunmethylated CpG motifs (“CpGs”, e.g., as available from ColeyPharmaceutical). In another embodiment, the immunostimulatory moleculeis a STING agonist. Such immunostimulatory agents may be administeredsimultaneously, separately or sequentially with the anti-LAP antibodiesor antigen binding fragments described herein.

Suitable immune checkpoint blockers include, but are not limited to,agents (e.g., antibodies) that bind to PD-1, PD-L1, PD-L2, LAG-3, CTLA4,TIGIT, ICOS, OX40, PVR, PVRIG, VISTA, and TIM3. Non-limiting examples ofantibodies that bind to PD-1, PD-L1, and PD-L2 include pembrolizumab;nivolumab; MK-3475; MPDL32; MEDI0680; MEDI4736; AMP-224; andMSB0010718C.

In some embodiments, the anti-LAP antibody or antigen binding fragmentis administered with an agent that targets a stimulatory or inhibitorymolecule that is a member of the immunoglobulin super family (IgSF). Forexample, the anti-LAP antibodies or antigen binding fragments describedherein, may be administered to a subject with an agent that targets amember of the IgSF family to increase an immune response. For example,an anti-LAP antibody or antigen binding fragment may be administeredwith an agent that targets a member of the B7 family of membrane-boundligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2(ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or a co-stimulatory orco-inhibitory receptor binding specifically to a B7 family member.

An anti-LAP antibody or antigen binding fragment may also beadministered with an agent that targets a member of the TNF and TNFRfamily of molecules (ligands or receptors), such as CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L,TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL,TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT,DcR3, HVEM, VEGETL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, Lymphotoxin α/TNFβ,TNFR2, TNFα, LTβR, Lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, andNGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

T cell responses can be stimulated by a combination of anti-LAPantibodies or antigen binding fragments described herein and one or moreof the following agents:

-   -   (1) An antagonist (inhibitor or blocking agent) of a protein        that inhibits T cell activation (e.g., immune checkpoint        inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as        described above, and any of the following proteins: TIM-3,        Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113,        CD155, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1,        TIM-1, and TIM-4; and/or    -   (2) An agonist of a protein that stimulates T cell activation,        such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS,        ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate the above proteins and may be combinedwith the anti-LAP antibodies or antigen binding fragments describedherein for treating cancer, include: Yervoy™ (ipilimumab) orTremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1),MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A(to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3),IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137),CDX-1127 (to CD27), anti-OX40 (Providence Health Services), huMAbOX40L(to OX40L), Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (toCD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3), Ipilumumab (toCTLA-4).

Other molecules that can be combined with anti-LAP antibodies or antigenbinding fragments for the treatment of cancer include antagonists ofinhibitory receptors on NK cells or agonists of activating receptors onNK cells. For example, anti-LAP antibodies or antigen binding fragmentscan be combined with antagonists of KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-LAPantibodies may be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In certain embodiments, anti-LAP antibodies or antigen binding fragmentscan be used in combination with (i) antagonists (or inhibitors orblocking agents) of proteins of the IgSF family or B7 family or the TNFfamily that inhibit T cell activation or antagonists of cytokines thatinhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF;“immunosuppressive cytokines”) and/or (ii) agonists of stimulatoryreceptors of the IgSF family, B7 family or the TNF family or ofcytokines that stimulate T cell activation, for stimulating an immuneresponse, e.g., for treating proliferative diseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155 (seePCT publication numbers WO11/70024, WO11/107553, WO11/131407,WO13/87699, WO13/119716, and WO13/132044) or FPA-008 (see PCTpublication numbers WO11/140249; WO13169264; and WO 14/036357).

Additional agents that may be combined with anti-LAP antibodies orantigen binding fragments include agents that enhance tumor antigenpresentation, e.g., dendritic cell vaccines, GM-CSF secreting cellularvaccines, CpG oligonucleotides, and imiquimod, or therapies that enhancethe immunogenicity of tumor cells (e.g., anthracydines).

Another therapy that may be combined with anti-LAP antibodies is atherapy that inhibits a metabolic enzyme such as indoleamine dioxygenase(IDO), tryptophan-2,3-dioxygenase, dioxygenase, arginase, or nitricoxide synthetase.

Another class of agents that may be used with anti-LAP antibodiesincludes agents that inhibit the formation of adenosine or inhibit theadenosine A2A receptor, for example, anti-CD73 antibodies, anti-CD39antibodies, and adenosine A2A/A2b inhibitors.

Other therapies that may be combined with anti-LAP antibodies or antigenbinding fragments for treating cancer include therapies thatreverse/prevent T cell anergy or exhaustion and therapies that triggeran innate immune activation and/or inflammation at a tumor site.

The anti-LAP antibodies or antigen binding fragments may be combinedwith a combinatorial approach that targets multiple elements of theimmune pathway, such as one or more of the following: a therapy thatenhances tumor antigen presentation (e.g., dendritic cell vaccine,GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); atherapy that inhibits negative immune regulation e.g., by inhibitingCTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blockingregulatory T cells or other immune suppressing cells; a therapy thatstimulates positive immune regulation, e.g., with agonists thatstimulate the CD-137 and/or GITR pathway and/or stimulate T celleffector function; a therapy that increases systemically the frequencyof anti-tumor T cells; a therapy that depletes or inhibits regulatory Tcells using an antagonist of CD25 (e.g., daclizumab) or by ex vivoanti-CD25 bead depletion; a therapy that impacts the function ofsuppressor myeloid cells in the tumor; a therapy that enhancesimmunogenicity of tumor cells (e.g., anthracyclines); cell therapy withadoptive T cell or NK cell transfer including genetically modifiedcells, e.g., cells modified by chimeric antigen receptors (CAR-Ttherapy); a therapy that inhibits a metabolic enzyme such as indoleaminedioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; atherapy that reverses/prevents T cell anergy or exhaustion; a therapythat triggers an innate immune activation and/or inflammation at a tumorsite; administration of immune stimulatory cytokines; or blocking ofimmunosuppressive or immunorepressive cytokines.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with proinflammatory cytokines, for example, IL-12 andIL-2. These cytokines can be modified to enhance half-life and tumortargeting.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with immune cell engagers such as NK cell engagers or Tcell engagers.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with indoleamine dioxygenase (IDO) inhibitors,tryptophan-2,3-dioxygenase (TDO) inhibitors, and dual IDO/TDOinhibitors.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with kynurine inhibitors.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with CD47 and/or SIRPa blocking therapies.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with JAK inhibitors and JAK pathway inhibitors (e.g.,STAT3 inhibitors), e.g., for the treatment of myelofibrosis andmyeloproliferative neoplasms.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with DNA damage repair inhibitors.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with erythropoietin and drugs that stimulatehematopoiesis.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with angiogenesis inhibitors.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with anti-viral drugs, such as neuramidase inhibitors.

Bispecific antibodies which have a first binding region with thespecificity of the anti-LAP antibodies or antigen binding fragmentsdescribed herein and a second binding region which binds to an immunecheckpoint blocker (e.g., PD-1, PD-L1) can be used in combination withat least one additional anti-cancer agent (e.g., radiation,chemotherapeutic agents, biologies, vaccines) to inhibit tumor growth.

The anti-LAP antibodies or antigen binding fragments described hereincan be combined with one or more immunostimulatory antibodies, such asan anti-PD-1 antagonist antibody, an anti-PD-L1 antagonist antibody, anantagonist anti-CTLA-4 antibody, an antagonistic anti-TIM3 antibody,and/or an anti-LAG3 antagonist antibody, such that an immune response isstimulated in the subject, for example to inhibit tumor growth.

Exemplary anti-PD-1 antibodies include nivolumab, pembrolizumab (alsoknown as MK-3475, Lambrolizumab) described in WO2012/145493; AMP-514described in WO 2012/145493, as well as PD-1 antibodies and other PD-1inhibitors described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO2011/066389, WO 2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757and 8,217,149, and U.S. Patent Publication No. 2009/0317368.

Exemplary anti-PD-L1 antibodies include MEDI4736 (also known asAnti-B7-H1), MPDL3280A (also known as RG7446), MSB0010718C(WO2013/79174), rHigM12B7, as well as any of the anti-PD-L1 antibodiesdisclosed in WO2013/173223, WO2011/066389, WO2012/145493, U.S. Pat. Nos.7,635,757 and 8,217,149 and U.S. Publication No. 2009/145493.

Exemplary anti-CTLA-4 antibodies include Yervoy™ (ipilimumab),tremelimumab (formerly ticilimumab, CP-675,206), or an anti-CTLA-4antibody described in any of the following publications: WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin.Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr etal. (1998) Cancer Res. 58:5301-5304.

Exemplary anti-LAG3 antibodies include IMP731 and IMP-321, described inUS Publication No. 2011/007023, and PCT publication numbers WO08/132601,and WO09/44273, as well as antibodies described in U.S. PatentPublication No. US2011/0150892, and international patent publicationnumbers WO10/19570 and WO2014/008218.

Anti-LAP antibodies or antigen binding fragments can also be combinedwith immune-oncology agents such as CD137 (4-1BB) agonists (e.g., anagonistic CD137 antibody such as urelumab or PF-05082566 (see PCTpublication number WO12/32433)); GITR agonists (e.g., an agonisticanti-GITR antibody), CD40 agonists (e.g., an agonistic CD40 antibody);CD40 antagonists (e.g., an antagonistic CD40 antibody such aslucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4);CD27 agonists (e.g., an agonistic CD27 antibody such as varlilumab(CDX-1127)), MGA271 (to B7H3) (WO 11/109400)); KIR antagonists (e.g.,lirilumab); IDO antagonists (e.g., INCB-024360 (WO2006/122150,WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620,WO09/1156652, WO11/56652, WO12/142237) or F001287); Toll-like receptoragonists (e.g., TLR2/4 agonists (e.g., Bacillus Calmette-Guerin); TLR7agonists (e.g., Hiltonol or Imiquimod); TLR7/8 agonists (e.g.,Resiquimod); or TLR9 agonists (e.g., CpG7909)); and TGF-β inhibitors(e.g., GC1008, LY2157299, TEW7197, or IMC-TR1).

The anti-LAP antibodies or antigen binding fragments described hereincan also be combined with an immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al. (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF (discussed further below).

The anti-LAP antibodies or antigen binding fragments described hereincan also be combined with an anti-neoplastic antibody, such as Rituxan®(rituximab), Herceptin® (trastuzumab), Bexxar® (tositumomab), Zevalin®(ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab),Avastin® (bevacizumab), and Tarceva® (erlotinib), and the like.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). Ex vivo activation in the presence of theanti-LAP antibodies described herein with or without an additionalimmunostimulating therapy (e.g., an immune checkpoint blocker) can beexpected to increase the frequency and activity of the adoptivelytransferred T cells.

The anti-LAP antibody or antigen binding fragment may also beadministered with a standard of care treatment, or another treatment,such as radiation, surgery, or chemotherapy.

The anti-LAP antibody or antigen binding fragment may be combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principlesand Practice of Oncology, Fifth Edition). In one of these strategies, avaccine is prepared using autologous or allogeneic tumor cells. Thesecellular vaccines have been shown to be most effective when the tumorcells are transduced to express GM-CSF. GM-CSF has been shown to be apotent activator of antigen presentation for tumor vaccination (Dranoffet al. (1993) Proc. Natl. Acad Sci U.S.A. 90: 3539-43).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withthe anti-LAP antibodies or antigen binding fragments described herein toactivate more potent anti-tumor responses.

In some embodiments, the combination of therapeutic antibodies discussedherein can be administered concurrently as a single composition in apharmaceutically acceptable carrier, or concurrently as separatecompositions with each antibody in a pharmaceutically acceptablecarrier. In another embodiment, the combination of therapeuticantibodies can be administered sequentially.

X. Kits

Also provided are kits comprising the anti-LAP antibodies or antigenbinding fragments, multispecific molecules, or immunoconjugatesdisclosed herein, optionally contained in a single vial or container,and include, e.g., instructions for use in treating or diagnosing adisease (e.g., cancer). The kits may include a label indicating theintended use of the contents of the kit. The term label includes anywriting, marketing materials or recorded material supplied on or withthe kit, or which otherwise accompanies the kit. Such kits may comprisethe antibody, multispecific molecule, or immunoconjugate in unit dosageform, such as in a single dose vial or a single dose pre-loaded syringe.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents, and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES

Commercially available reagents referred to in the Examples below wereused according to manufacturer's instructions unless otherwiseindicated. Unless otherwise noted, the present invention uses standardprocedures of recombinant DNA technology, such as those describedhereinabove and in the following textbooks: Sambrook et al., supra;Ausubel el al., Current Protocols in Molecular Biology (Green PublishingAssociates and Wiley Interscience, N.Y., 1989); Innis et al, PCRProtocols; A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al., Antibodies; A Laboratory Manual (Cold SpringHarbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis(IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coliganet al., Current Protocols in Immunology, 1991.

The following Examples describe the characterization of anti-LAPantibodies 28G11, 22F9, 20E6 (also referred to as 26E10), 17G8, and24E3, which were generated by immunizing TGFβ1 knock-out mice with mouseTGFβ1, as described in Oida et al. (PLoS One 2010; 5(11):e15523). TheCDR sequences, variable region sequences, and full-length heavy andlight chain sequences of anti-LAP antibodies 28G11, 22F9, 20E6, 17G8,and 24E3 are provided in Table 34. These antibodies were prepared inmurine antibody format with an mIgG2a constant region, chimeric formatwith a human IgG constant region, and/or in humanized format.

To generate the antibodies with mIgG2a constant regions, the variableregion sequences of each antibody were fused to a murine IgG2a constantregion. The murine V_(H) domains were fused to a codon-optimized genefor the murine IgG2a constant domains (UniProt accession #P01863) usingoverlap extension PCR. The murine V_(L) domains were fused to acodon-optimized gene for the murine kappa constant domain (UniProtaccession #P01837) using overlap extension PCR. The complete heavy-chainand light-chain sequences were individually TOPO-TA cloned into pcDNA3.4for expression in ExpiCHO cells.

To generate the antibodies in chimeric format, the variable regionsequences of the murine parental clones were fused to the human IgG1constant region sequences. The murine VH domains were fused to acodon-optimized gene for the human IgG1 constant domains (UniProtaccession #P01857) using overlap extension PCR. The murine VL domainswere fused to a codon-optimized gene for the human kappa constant domain(UniProt accession #P01834) using overlap extension PCR. The completeheavy-chain and light-chain sequences were individually TOPO-TA clonedinto pcDNA3.4 for expression in ExpiCHO cells.

Details regarding the humanization of the antibodies are described inExamples 8-11.

The designation of antibodies will follow the format described in Table2.

TABLE 2 Designation Description Antibody clone_(hyb) Parental murineantibody (e.g., 28G11_(hyb)) Antibody_mIgG2a Murine variable region ofparental antibody fused (e.g., 28G11_IgG2a) to murine IgG2a constantregion Antibody_hIgG1 (e.g., Chimeric antibody with murine variableregion of 28G11_hIgG1) parental antibody and human IgG1 constant regionAntibody_H(X)L(Y) Humanized antibody with X referring to particular(e.g., 28G11_H2L3) humanized heavy chain and Y referring to particularhumanized light chain

Example 1: Binding of Anti-LAP Antibodies to Human and Murine LAP-TGFβ1

This Example describes the ability of anti-LAP antibodies 28G11_hIgG1,22F9_hIgG1, and 20E6_hIgG1 to bind to human and murine LAP-TGFβ1 usingbio-layer interferometry.

The chimeric antibodies were biotinylated using EZ-LinkSulfoNHS-LC-Biotin (ThermoFisher). A streptavidin-functionalized tip wasequilibrated in binding buffer (10 mM sodium phosphate, 150 mM sodiumchloride, 1% (w/v) bovine serum albumin, 0.05% (w/v) sodium azide, pH7.4). The tip was dipped in a 10 μg/mL solution of biotinylated,chimeric anti-LAP in binding buffer for 15 seconds to load the tip withantibody. The antibody-loaded tip was then washed in binding buffer andplaced in a solution containing 0-24 nM of LAP-TGFβ1 (either a fusionprotein containing a human IgG1 Fc domain fused to human LAP-TGFβ1 or amurine LAP-TGFβ1 with a C-terminal polyhistidine purification tag). Theantigen was allowed to bind to antibody for 5 minutes (associationphase), and then the tip was moved to binding buffer (dissociationphase). The association and dissociation phases were fit to a 1:1binding model to determine the binding rate constants.

As shown in Table 3, 28G11_hIgG1, 22F9_hIgG1, and 20E6_hIgG1 bind withsub-nanomolar affinity to both human and murine LAP-TGFβ1. These datademonstrate that the antibodies bind to human and murine LAP-TGFβ1 inthe absence of an anchor protein.

TABLE 3 Antibody k_(on) (×10⁶ M⁻¹s⁻¹ k_(off) (×10⁴ s⁻¹) K_(D) (nM) MouseLAP-TGFβ1 28G11_hIgG1 0.84 1.58 0.19 22F9_hIgG1 1.21 4.44 0.3720E6_hIgG1 1.39 5.01 0.36 Human LAP-TGFβ1 28G11_hIgG1 0.54 3.60 0.6722F9_hIgG1 1.01 7.68 0.76 20E6_hIgG1 1.04 6.06 0.58

Example 2: Binding of Anti-LAP Antibodies to LAP-TGFβ Isoforms andLAP-TGFβ Variants

This Example describes the binding of anti-LAP antibodies to LAP-TGFβisoforms and LAP-TGFβ variants. In addition to antibodies 28G11_hIgG1,22F9_hIgG1, and 20E6_hIgG1, anti-LAP antibodies 17G8_hIgG1, 24E3_hIgG1,and 2C9_(hyb) were also tested in this experiment. Briefly, 4×10⁵ eachof (a) HT1080 cells, (b) HT1080 cells overexpressing human LAP-TGFβ1,(c) HT1080 cells overexpressing human LAP-TGFβ2, (d) HT1080 cellsoverexpressing human LAP-TGFβ3, (e) HT1080 cells overexpressing murineLAP-TGFβ1, (f) P3U1 cells, (g) P3U1 cells overexpressing LAP-TGFβ1 andGARP, and (h) P3U1 cells overexpressing LAP-TGFβ1 and LRRC33 werecultured in 96-well plates. The plates were centrifuged for 5 min at1,500 rpm, liquid was removed, and cells were resuspended with 200 μLFACS buffer. The plates were centrifuged again, diluted primary antibodywas added to each well, and the plates were incubated on ice for 20minutes, followed by centrifugation. The cells were resuspended in 200μL FACS buffer, centrifuged again, and resuspended in 50 μL dilutedsecondary antibody (Alexa647-anti-Human IgG or APC-anti-Mouse IgG). Theplates were incubated on ice for 20 minutes in the dark, washed twicewith 200 μL FACS buffer, and cells from each well (in 200 μL FACSbuffer) were read on the Attune NXT instrument.

As shown in FIGS. 1A-1F, all tested antibodies bind to HT1080 cell linesoverexpressing human LAP-TGFβ1, but not to control HT1080 cells or cellsoverexpressing human LAP-TGFβ2 or LAP-TGFβ3. All tested antibodies bindto P3U1-hTGFβ1 cells, and the binding was enhanced when either humanGARP or LRRC33 was co-expressed. Antibodies 28G11_hIgG1, 22F9_hIgG1,20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1, but not 2C9_(hyb), bind toHT1080 cells overexpressing mouse LAP-TGFβ1. These results indicate thatantibodies 28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and24E3_hIgG1 bind specifically to the LAP-TGFβ1 isoform of TGFβ.

The ability of the anti-LAP antibodies to bind variants of TGFβ1 thateither prevent TGFβ1 activation by integrins (“closed” conformation) orfavor release (“open” conformation), chimeric TGFβ1 sequences containingresidues from chicken TGFβ1, and the LAP-only TGFβ1 variant (i.e., humanTGFβ1 variant which does not contain the mature cytokine) was tested.

Briefly, 4×10⁵ each of (a) HT1080 cells, (b) HT1080 cells overexpressinghuman LAP-TGFβ1, (c) HT1080 cells overexpressing LAP-TGFβ1 with K27C andY75C mutations, (d) HT1080 cells overexpressing LAP-TGFβ1 with a Y74Tmutation, (e) HT1080 cells overexpressing chimeric LAP-TGFβ1 in whichexon 2.3 (residues 131-164) of human LAP-TGFβ1 have been replaced withcorresponding residues from chicken LAP-TGFβ1 (UniProt accession#H9CX01), (f) HT1080 cells overexpressing chimeric LAP-TGFβ1 in whichexon 4 (residues 183-208) of human LAP-TGFβ1 has been replaced with exon4 from chicken LAP-TGFβ1, (g) HT1080 cells overexpressing chimericLAP-TGFβ1 in which exon 2.2 (residues 108-130) of human LAP-TGFβ1 hasbeen replaced with exon 2.2 from chicken LAP-TGFβ1, and (h) HT1080 cellsoverexpressing the LAP-only variant (i.e., “empty LAP”) were cultured in96-well plates. Cells were processed for flow cytometry in the samemanner described above for the isoform-specific binding experiments.

As shown in FIGS. 2A-2F, while none of the tested anti-LAP antibodiesbind to untransduced HT1080 cells, all tested antibodies bind to HT1080cells overexpressing wild-type human LAP-TGFβ1. Antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 bind to the K27C/Y75C(“closed”) LAP-TGFβ1 variant, but not to the Y74T (“open”) LAP-TGFβ1variant. In contrast, antibody 2C9_(hyb) binds to both the K27C/Y75C andY74T LAP-TGFβ1 variants. Antibodies 28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1,17G8_hIgG1, and 24E3_hIgG1, but not 2C9_(hyb), bind to chimericLAP-TGFβ1 containing chicken exons #2.3 and #4. Furthermore, as shown inFIGS. 3A-3F, while all tested antibodies bind to HT1080 cellsoverexpressing wild-type human LAP-TGFβ1, antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 did not bind to theLAP-only variant or to chimeric LAP-TGFβ1 containing chicken exon #2.2.

To determine whether anti-LAP antibodies 28G11_hIgG1, 22F9_hIgG1,20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1, bind to free human TGFβ1 (i.e.,mature TGFβ1 that lacks LAP), the ability of the antibodies to inhibitan anti-TGFβ ELISA was evaluated. Briefly, mature TGFβ1 (1000 pg) wasincubated with the indicated anti-LAP antibodies at 10 μg/mL, an isotypecontrol antibody as a negative control or the commercially availableanti-TGFβ antibody 1D11 as a positive control for 10 minutes on ice. Forexample, the 1D11 antibody is commercially available from any number ofsellers, for example Bio x Cell Inc. (West Lebanon, N.H.). Supernatantswere assayed in a TGFβ1 ELISA (R&D Systems) according to themanufacturer instructions to measure free TGFβ1. As shown in FIG. 4, theanti-TGFβ antibody 1D11 bound to the mature TGFβ and inhibited theELISA, while no inhibition was seen with 28G11_hIgG1, 22F9_hIgG1,20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1. These data demonstrate that28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1antibodies do not bind to mature TGFβ1 that lacks LAP. Note that Figureand FIG. are used interchangeably in this application.

In summary, these results suggest that antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 share a relatedepitope. All anti-LAP antibodies tested bind to HT1080 cellsoverexpressing wild-type human LAP-TGFβ1 (HT1080-hβ1), but not tountransduced HT1080 cells. Antibodies 28G11_hIgG1, 22F9_hIgG1,20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 did not bind to the LAP-onlyconstruct or the chimera containing chicken exon #2.2. Antibody2C9_(hyb) binds to both the LAP-only construct, but not to the exon #2.2chimera. This compilation of data indicates that antibodies 28G11_hIgG1,22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 only bind to LAP thatcontains the mature cytokine, and this is supported by the results shownin FIG. 4. However, chimeras with chicken exons #6 and #7, whichencompass the mature cytokine, were bound by 28G11_hIgG1 (data notshown), initially suggesting that 28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1,17G8_hIgG1, and 24E3_hIgG1 do not bind the mature cytokine directly, butrather are sensitive to conformational changes in the LAP region inducedby the presence or absence of the mature cytokine. In contrast, 2C9binds to all variants of LAP, including the “open” and “closed”conformation variants, as well as LAP in the presence or absence of themature cytokine.

The binding epitope for 28G11 was mapped by assessing binding ofcell-surface chimeric human/chicken LAP-TGFβ1 molecules using flowcytometry. The use of these binding data to determine epitopes was basedon the following assumptions:

-   -   1. Anti-LAP antibodies will not bind to chicken LAP-TGFβ        sequence    -   2. Human/chicken chimeras are expressed and displayed properly        on the cell surface    -   3. If an anti-LAP antibody does bind to a chimera, none of the        residues in that exon are part of the epitope.    -   4. If an anti-LAP antibody does not bind to a chimera, at least        one residue in that exon is part of the epitope, or the presence        of chicken sequence in that exon causes a conformational change        in another part of LAP-TGFβ that disrupts the epitope.

Chimeric LAP-TGFβ1 molecules were made by replacing individual exons inhuman LAP-TGFβ1 with the homologous chicken LAP-TGFβ1 sequence (Table4). Human and chicken LAP-TGFβ1 share ˜50% sequence identity. The aminoacid sequences of the tested LAP-TGFβ1 sequences are shown in Table 34.

TABLE 4 LAP-TGFβ1 sequences used for epitope mapping SEQ ID NameDescription 1 human Wild-type human LAP-TGFβ1 sequence LAP-TGFβ1 (huB1)198 chicken Wild-type chicken LAP-TGFβ1 sequence LAP-TGFβ1 (chB1) 199chimera #1 chimeric LAP-TGFβ1 in which exon 1 (residues 1-89) of humanLAP-TGFβ1 has been replaced with exon 1 from chicken LAP-TGFβ1 200chimera #1.2 chimeric LAP-TGFβ1 in which exon 1.2 (residues 30-50) ofhuman LAP-TGFβ1 has been replaced with exon 1.2 from chicken LAP-TGFβ1201 chimera #1.3 chimeric LAP-TGFβ1 in which exon 1.3 (residues 51-81)of human LAP-TGFβ1 has been replaced with exon 1.3 from chickenLAP-TGFβ1 202 chimera #2 chimeric LAP-TGFβ1 in which exon 2 (residues90-143) of human LAP-TGFβ1 has been replaced with exon 2 from chickenLAP-TGFβ1 203 chimera #2.1 chimeric LAP-TGFβ1 in which exon 2.1(residues 82-107) of human LAP-TGFβ1 has been replaced with exon 2.1from chicken LAP-TGFβ1 204 chimera #2.2 chimeric LAP-TGFβ1 in which exon2.2 (residues 108-130) of human LAP-TGFβ1 has been replaced with exon2.2 from chicken LAP-TGFβ1 205 chimera #2.3 chimeric LAP-TGFβ1 in whichexon 2.3 (residues 131-164) of human LAP-TGFβ1 have been replaced withcorresponding residues from chicken LAP-TGFβ1 206 chimera #3 chimericLAP-TGFβ1 in which exon 3 (residues 144-182) of human LAP-TGFβ1 havebeen replaced with corresponding residues from chicken LAP-TGFβ1 207chimera #4 chimeric LAP-TGFβ1 in which exon 4 (residues 183-208) ofhuman LAP-TGFβ1 has been replaced with exon 4 from chicken LAP-TGFβ1 208chimera #5 chimeric LAP-TGFβ1 in which exon 5 (residues 209-257) ofhuman LAP-TGFβ1 have been replaced with corresponding residues fromchicken LAP-TGFβ1 209 chimera #6 chimeric LAP-TGFβ1 in which exon 6(residues 258-309) of human LAP-TGFβ1 have been replaced withcorresponding residues from chicken LAP-TGFβ1 210 chimera #7 chimericLAP-TGFβ1 in which exon 7 (residues 310-361) of human LAP-TGFβ1 havebeen replaced with corresponding residues from chicken LAP-TGFβ1

These constructs were subcloned into lentivirus and transduced intoHT1080 cells. Successful gene integration was confirmed by expression ofa green fluorescent protein (GFP) reporter gene. LAP-TGFβ1 expressionwas evaluated by flow cytometry using a rabbit monoclonal antibody(Rmab) raised against a peptide from the mature cytokine (residues250-361) domain (Abeam cat #ab179695), 28G11_(hyb) and 2C9_(hyb).Briefly, 4×10⁵ each of (a) HT1080 cells, (b) HT1080 cells overexpressinghuman LAP-TGFβ1, (c) HT1080 cells overexpressing chicken LAP-TGFβ1, and(d) HT1080 cells overexpressing human/chicken chimeras #1-#7 werecultured in 96-well plates. The plates were centrifuged for 5 min at1,500 rpm, liquid was removed, and cells were resuspended with 200 μLFACS buffer. The plates were centrifuged again, diluted primary antibodywas added to each well, and the plates were incubated on ice for 20minutes, followed by centrifugation. The cells were resuspended in 200μL FACS buffer, centrifuged again, and resuspended in 50 μL dilutedsecondary antibody (APC-anti-Mouse IgG). The plates were incubated onice for 20 minutes in the dark, washed twice with 200 μL FACS buffer,and cells from each well (in 200 μL FACS buffer) were read on the AttuneNXT instrument. The antibodies were considered to be binding if >10% ofcells were GFP+/APC+ and there was visible correlation between the GFPand allophycocyanin (APC) signals (Table 5).

TABLE 5 % GFP+/APC+ cells by flow cytometry HT1080 Variant Rmab 28G11(hyb) 2C9 (hyb) null  0.7%  0.0%  0.0% huB1 68.9% 98.3% 72.4% chB1  0.5% 0.3%  1.1% chimera #1  7.5%  0.2%  7.6% chimera #1.2 78.3% 84.1% 81.8%chimera #1.3 65.2% 83.0% 82.6% chimera #2  1.5%  0.2%  4.1% chimera #2.179.1%  3.5%  5.6% chimera #2.2 47.2%  0.5%  0.2% chimera #2.3 ND 94.7% 0.3% chimera #3 46.3% 32.2% 34.0% chimera #4 56.2% 76.2%  2.8% chimera#5 11.9% 41.7%  4.0% chimera #6 76.7% 84.9% 87.6% chimera #7  0.6% 44.4%63.9%

All three antibodies bound to the positive control HT1080 cell lineoverexpressing human LAP-TGFβ1. No binding was observed with thenegative control strain (HT 1080-null). None of the antibodies boundchicken LAP-TGFβ1, indicating that the sequence differences betweenhuman and chicken homologues were sufficient to disrupt the epitopesrecognized by these antibodies. Chimeras #1 and #2 were not recognizedby any antibody, suggesting that these constructs were not efficientlyexpressed. To test this hypothesis, smaller portions of chicken sequencewere inserted into exon #1 or #2 in the human sequence (chimeras #1.2,1.3, 2.1, 2.2, and 2.3). The constructs with these smaller replacementswere recognized by the Rmab antibody, indicating that they were robustlyexpressed in HT1080 cells. Chimera #7 was not bound by the Rmabantibody, but was recognized by both 28G11_(hyb) and 2C9_(hyb), showingthat this construct is expressed on the cell surface, and that theepitope for Rmab is likely in this region of the protein.

The 28G11_(hyb) antibody bound to chimeras #1.2, 1.3, 2.3, 3, 4, 5, 6and 7, indicating that those regions are not involved in the epitope forthis antibody. In contrast, chimeras #2.1 and #2.2 were not bound by28G11 (hyb), suggesting that this antibody binds to human LAP-TGFβ1within the sequence VLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAE (SEQ IDNO: 215).

The 2C9_(hyb) binding was disrupted by the chicken residues present inchimeras #2.1, 2.2, 2.3, 4 and 5. This suggests that this antibody bindsto a discontinuous epitope that incorporates portions of each of theseinsertions that are distant in sequence, but adjacent in thethree-dimensional structure of the antigen.

Example 3. Generation of Antibodies Binding to Specific Epitopes ofInterest on LAP-TGFβ1

As described in Example 2, constructs with combinations of human andchicken sequence are able to fold into the correct structure.Accordingly, additional chimeras can be generated that could be used asimmunogens to target specific epitopes of interest on LAP-TGFβ1. Theseconstructs would be the inverse of the constructs described in Example2. That is, the majority of the sequence would be taken from chickenLAP-TGFβ1, with small sections of human LAP-TGFβ1 inserted in regionscontaining the desired epitope. Exemplary epitopes on LAP-TGFβ1 that canbe targeted using this strategy include, for example, an epitopecomprising amino acids 82-130 of human LAP-TGFβ1, the lower arm ofLAP-TGFβ1, or the latency loop of LAP-TGFβ1. This chimeric protein couldbe used to immunize chickens to yield monoclonal antibodies. Since thechicken LAP-TGFβ1 would be recognized as self, the immune response willbe focused on the human sequence. Exemplary chicken-human chimeras whichcan be used to immunize chickens is shown in Table 6. The sequences ofthese chimeras are provided in Table 34. Anti-LAP antibodies generatedin this manner can be tested for various functions (e.g., binding tohuman LAP-TGFβ1, inhibition of TGFβ1 activation, binding to immunecells) using the methods described herein.

TABLE 6 Chicken-human chimera for immunization SEQ ID Name Description211 chB1ex2.1_2.2 Chimeric LAP-TGFβ1 in which exons 2.1 and 2.2(residues 83-128) of chicken LAP-TGFβ1 has been replaced with exons 2.1and 2.2 from human LAP-TGFβ1. This chimera can be used to generateantibodies that target amino acid residues 82-130 of human LAP-TGFβ1.212 chB1ex1.3 Chimeric LAP-TGFβ1 in which exon 1.3 (residues 50-82) ofchicken LAP-TGFβ1 has been replaced with exon 1.3 from human LAP- TGFβ1.This chimera can be used to generate antibodies that target amino acidresidues 50-81 of human LAP-TGFβ1 213 chB1_ex1.2 Chimeric LAP-TGFβ1 inwhich exon 1.2 (residues 30-50) of chicken LAP-TGFβ1 has been replacedwith exon 1.2 from human LAP- TGFβ1. This chimera can be used togenerate antibodies that target amino acid residues 30-50 of humanLAP-TGFβ1

Example 4: Effect of Anti-LAP Antibodies on TGFβ1 Activation

This Example describes the effect of anti-LAP antibodies 28G11_hIgG1,28G11_mIgG2a, 17G8_hIgG1, 24E3_hIgG1, 22F9_hIgG1, 22F9_mIgG2a,20E6_hIgG1, and 20E6_mIgG2a antibodies on TGFβ1 activation.

Briefly, P3U1 cells expressing human LAP-TGFβ1 or P3U1 cells expressingmurine LAP-TGFβ1 were cultured overnight in serum free advanced DMEM inround bottom tissue culture plates and treated the following day withanti-LAP antibody (2-fold serial dilutions starting with a high of 20ug/ml) for 24 hours. Active TGFβ1 was detected in the supernatant ofcell cultures by utilizing a commercially available human TGFβ1 ELISAkit (R&D Systems) according to the manufacturer instructions.

FIGS. 5A and 5B show that 28G11_hIgG1 inhibited TGFβ1 activation in bothP3U1 cells expressing human LAP-TGFβ1 (IC50=1.4 ug/ml) and murineLAP-TGFβ (IC50=1 ug/ml).

As with the experiment using 28G11 in FIG. 5, the tested antibodiesinhibited TGFβ1 activation in P3U1 cells expressing human LAP-TGFβ1.Data are shown in the following Figures; IC50 values are as shown forantibody 17G8 (FIGS. 6A for human TGFβ1 (IC50=1.5 ug/ml) and 6B formouse TGFβ1 (IC50=0.59 ug/ml)), antibody 24E3 (FIGS. 6C for human TGFβ1(IC50=2.6 ug/ml) and 6D for mouse TGFβ1 (IC50=0.76 ug/ml)), antibody22F9 (FIG. 6E for human TGFβ1 (IC50=1.8 ug/ml)), and antibody 20E6 (FIG.6F for human TGFβ1 (IC50=1.08 ug/ml)).

Additional experiments were performed to further confirm that theantibodies inhibited activation in P3U1 cells expressing human LAP-TGFβ1or P3U1 cells expressing murine LAP-TGFβ1. Data are shown in thefollowing Figures; IC50 values are as shown: antibody 28G11_mIgG2a (FIG.6G: IC50=1.6 ug/ml for human TGFβ1 and 0.8 ug/ml for mouse TGFβ1),antibody 20E6_mIgG2a (FIG. 6H: IC50=1.4 ug/ml for human TGFβ1 and 1.0ug/ml for mouse TGFβ1), antibody 22F9_mIgG2a (FIG. 6I: IC50=1.8 ug/mlfor human TGFβ1 and 1.0 ug/ml for mouse TGFβ1), antibody 24E3_hIgG1(FIG. 6J: IC50=2.5 ug/ml for human TGFβ1 and 1.1 ug/ml for mouse TGFβ1),antibody 17G8_hIgG1 (FIG. 6K: IC50=1.0/ml for human TGFβ1 and 1.4 ug/mlfor mouse TGFβ1), antibody 20E6_H0.2aL1 (FIG. 6L: IC50=1.3 ug/ml forhuman TGFβ1 and 1.7 ug/ml for mouse TGFβ1).

Consistent with the above results, in a separate experiment using theP3U1 cell-based assay, 28G11_hIgG1, 22F9_hIgG1, and 20E6_hIgG1 potentlyinhibited human TGFβ1 activation (Table 7).

TABLE 7 Inhibition of human TGFβ1 activation (IC₅₀) 28G11_hIgG122F9_hIgG1 20E6_hIgG1 2.3 μg/mL 3.9 μg/mL 4.7 μg/mL

Thus multiple experiments described in this example showed that theanti-LAP antibodies described herein had an inhibitory effect on TGFβ1activation.

Example 5: Binding of Anti-LAP Antibodies to Extracellular Matrix

This Example describes the ability of 28G11_hIgG1, 22F9_hIgG1,20E6_hIgG1, 2C9_mIgG2a, 16B4_mIgG2a, 17G8_hIgG1, and 24E3_hIgG1antibodies to bind LAP-TGFβ1 in ECM.

Briefly, to evaluate antibody binding to ECM, P3U1 cells were incubatedin round bottom tissue culture plates for 48 hours. Cells were thenremoved, leaving behind ECM on the surface of the plates. Threedifferent groups were compared: (a) P3U1 cells expressing humanLAP-TGFβ1, (b) P3E11 cells expressing murine LAP-TGFβ1, and (c) P3E11cells without LAP-TGFβ1 (null cells). Binding of antibody toLAP-TGFβ1/ECM was then determined using biotinylated anti-LAP antibodiesfollowed by incubation with streptavidin horseradish peroxidase (HRP)and 3′,5,5′-tetramethylbenzidine (TMB) substrate.

As shown in FIG. 7, 28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and24E3_hIgG1 antibodies did not bind to LAP-TGFβ1/ECM. In contrast,anti-LAP antibody 16B4 showed strong binding to murine LAP-TGFβ1 inLAP-TGFβ1/ECM and anti-LAP antibody 2C9 showed strong binding to humanLAP-TGFβ1 in LAP-TGFβ1/ECM. This result suggests that, while28G11_hIgG1, 22F9_hIgG1, 20E6_hIgG1, 17G8_hIgG1, and 24E3_hIgG1 bindstrongly to cells expressing murine or human LAP-TGFβ1, they did notbind to murine or human LAP-TGFβ1 in ECM.

Example 6: Binding of Anti-LAP Antibodies to Platelets

This Example describes the binding of anti-LAP antibodies to plateletsand their effects on platelet degranulation.

Briefly, a direct platelet binding assay was performed by flowcytometry. Diluted whole human blood was incubated with the indicatedconcentrations of directly conjugated anti-LAP antibodies (28G11_(hyb),20E6_mIgG2a, 22F9_mIgG2a, 17G8_hIgG1, and 24E3_hIgG1) for 15 minutes.The reactions were then incubated for an additional 15 minutes with acommercially available directly conjugated antibody against CD61(BioLegend), and analyzed by flow cytometry. The data represents theanti-LAP mean fluorescence intensity of CD61 positive platelets. Asshown in FIG. 8, anti-LAP antibodies 28G11, 20E6, 22F9, 17G8, and 24E3showed binding to platelets in a dose-responsive manner.

These anti-LAP antibodies were further tested for plateletdegranulation. Briefly, diluted whole human blood was incubated with theindicated concentrations of anti-LAP antibodies or adenosine diphosphate(ADP) as a positive control for 15 minutes. The reactions were thenincubated for an additional 15 minutes with directly conjugatedantibodies against CD61, to detect whole blood platelets, and CD62P(BioLegend) to detect degranulated platelets. The samples were analyzedby flow cytometry to determine the percentage of CD62P+ platelets.

As shown in FIGS. 9A-9E, none of the tested antibodies, i.e., 28G11,20E6, 22F9, 17G8, and 24E3, induced significant platelet degranulation,even at the highest dose tested.

Example 7: Differential Binding of Anti-LAP Antibodies to Immune Cells

This Example describes the binding of anti-LAP antibodies to differenttypes of immune cells.

Anti-LAP antibodies were tested for their ability to bind to THP-1cells, a cell line derived from a patient with acute monocytic leukemiathat is reported to express LRRC33. THP-1 cells were incubated with FACSbuffer and human Fc block followed by incubation with varyingconcentrations of Alexa 647 conjugated 28G11_(hyb), 22F9_mIgG2a,20E6_mIgG2a, 17G8_hIgG1, 24E3_hIgG1, 2C9_mIgG2a, or mIgG2a isotypecontrol. Cells were analyzed by flow cytometry and graphed as percentpositive THP-1 cells or median fluorescent intensity (MFI) of anti-LAPbinding. As shown in FIGS. 10A and 10B, antibodies 22F9, 17G8, 24E3,20E6, and 2C9 display strong binding to THP-1 cells. No binding abovebackground was seen for anti-LAP antibody 28G11. In another experiment,the binding of 20E6 mIgG2a and 7H4 hyb to THP-1 cells was compared usingthe methods described above. As shown in FIG. 10C, and consistent withFIGS. 10A and 10B, 20E6 showed strong binding to THP-1 cells, whereas7H4 showed no binding. In a separate experiment THP-1 cells wereincubated with FACS buffer and human Fc block followed by incubationwith 5 ug/ml of Alexa 647 conjugated 28G11_hyb, 22F9_mIgG2a,20E6_mIgG2a, 2C9_mIgG2a, or IgG2a isotype control. Cells were analyzedby flow cytometry and gated as single cells. Representative dot plotsare shown in FIG. 10D; in these plots, antibodies were at 5 ug/ml.

Anti-LAP antibodies were tested for their ability to bind to U937 cells,a myeloid cell line derived from a patient with histiocytic lymphoma.U937 cells were incubated with FACS buffer and human Fc block followedby incubation with varying concentrations of Alexa 647 conjugated28G11_hyb, 22F9_mIgG2a, or 20E6_mIgG2a. In a separate experiment U937cells were incubated with FACS buffer and human Fc block followed byincubation with 10 ug/ml of Alexa 647 conjugated 28G11_hyb, 22F9_mIgG2a,20E6_mIgG2a or mIgG2a isotype control (FIGS. 10E and 10F).Representative dot plots are shown in FIG. 10G; in these plots,antibodies were at 10 ug/ml. The anti-LAP antibodies 28G11, 22F9, and20E6 were shown to bind similarly to the U937 cells, both by MFI and thedose response of binding.

These data demonstrate that the anti-LAP antibodies 28G11, 22F9, and20E6 display comparable binding to one LAP+ myeloid cell line butdramatically different binding to another LAP+ myeloid cell line. Thefollowing experiments were performed to determine whether that samedifferential binding could be observed in non-transformed cellpopulations.

Anti-LAP antibodies were tested for their ability to bind to immunecells isolated from mice carrying CT26 tumors. Briefly, 1×10⁶ CT26 cellswere injected into the flank region of 6 male Balb/C mice. When meantumor volumes reached about 80 mm³, mice were treated with either IgG2aor IgG1 isotype control antibodies at 10 mg/kg (this was originally partof an experiment in which mice were treated with therapeutic antibodiesand these animals were intended to serve as controls). Mice were treatedagain 3 days later and harvested 7 days post first injection. Tumortissue was disassociated in a GentIMACS dissociator and digested withCollagenase IV/DNase1, strained through a 70-μm cell strainer andcounted. Spleen tissue was dissociated by passing through a 70-μm cellstrainer and counted. Cells were analyzed by flow cytometry using thefollowing scheme: Gate on live cells→Gate on single cells→Gate on CD45+cells→Gate on CD41-population→Gate on appropriate immune cell subsets asfollows:

-   -   CD4 T cells—CD45+, CD3+, CD4+    -   Regulatory T cells—CD45+, CD3+, CD4+, Foxp3+    -   CD8 T cells—CD45+, CD3+, CD8+    -   CD11b—CD45+, CD11b+    -   M2 macrophages—CD45+, CD11b+, F4/80+, CD206+    -   Dendritic cells—CD45+, F4/80−, CD11c+    -   M-MDSC—CD45+, CD11b+, F4/80−, Ly6G−, Ly6C high    -   G-MDSC—CD45+, CD11b+, F4/80−, Ly6G+    -   M1 macrophages—CD45+, CD11b+, F4/80+, MHC II high, CD206−    -   NK cells—CD45+, CD49b+

Binding of the anti-LAP antibodies was analyzed using Alexa 647-labeled28G11-IgG2a, 22F9-IgG2a, and 20E6-IgG2a. A summary of the data is shownin Table 8 and in FIG. 11. The three anti-LAP antibodies 28G11, 22F9 and20E6 display very different binding profiles to clinically relevantimmune cell subsets in tumor tissue. Most notably, 22F9 and 20E6 bind toa higher percentage of regulatory T cells, M2 macrophages, and M-MDSCscompared to 28G11. These data indicate that, although these threeantibodies all have similarities in their binding and functionalproperties (see Examples 1, 2, and 4-6), the antibodies display largedifferences in their binding to cell populations known to be importantin the immunosuppressive tumor microenvironment. For example, both 22F9and 20E6 bind a higher percentage of regulatory T cells, M2 macrophages,and M-MDSC than does 28G11. This supports the superiority of 22F9 and20E6 over 28G11 for the modulation of these important immunosuppressivecell populations in tumors. Moreover, the differences in binding of 22F9and 20E6 support the potential preferential use of one or the otherantibody in a given tumor depending on the makeup of the infiltratingleukocyte population.

TABLE 8 Binding to immune cells in tumor (expressed as % positive ± SEMof the 6 individual mice) Immune cell type 28G11 22F9 20E6 CD4 5.1 ± 1.044.2 ± 2.0 66.8 ± 2.4 Regulatory T cells 24.8 ± 3.0  46.7 ± 2.2 75.8 ±3.9 CD8 0.5 ± 0.1 29.8 ± 7.4  3.4 ± 0.9 CD11b 16.8 ± 2.3  70.2 ± 3.250.7 ± 5.6 M2 macrophages 8.0 ± 1.4 92.5 ± 0.5 47.2 ± 4.5 Dendriticcells 7.8 ± 1.3 50.4 ± 4.0 27.6 ± 4.5 M-MDSC 6.9 ± 0.8 64.0 ± 3.2 30.3 ±4.9 G-MDSC 2.4 ± 0.6 21.9 ± 2.1  7.0 ± 1.9

Splenocytes from the same mice were analyzed by flow cytometry inparallel with the tumor tissue. As shown in Table 9, antibodies 28G11,22F9, and 20E6 bind a lower percentage of immune cells in the spleen oftumor bearing mice than they do of the tumors from those same mice(Table 8) These data demonstrate the tumor selectivity of all threeantibodies. Some differences between the antibodies were observed, with22F9 displaying the strongest binding in the spleen. This supports thesuperiority of 20E6 in settings where a maximal selectivity for thetumor environment is preferred.

TABLE 9 Binding to immune cells in spleen (expressed as % positive ± SEMof the 6 individual mice) Immune cell type 28G11 22F9 20E6 CD4  1.6 ±0.05  2.9 ± 0.06  1.7 ± 0.06 Regulatory T cells 4.0 ± 0.4 6.1 ± 0.5 3.6± 0.3 CD8  0.9 ± 0.02 1.9 ± 0.1  0.9 ± 0.05 CD11b 4.0 ± 0.5 17.2 ± 1.1 4.2 ± 0.2 M2 macrophages 1.8 ± 0.6 11.3 ± 1.7  2.6 ± 0.5 Dendritic cells4.9 ± 0.5 21.5 ± 1.1  6.3 ± 0.5 M-MDSC 8.2 ± 0.6 21.7 ± 2.2  4.9 ± 0.3G-MDSC 1.6 ± 0.4 8.0 ± 0.4 2.1 ± 0.2 M1 macrophages 1.0 ± 0.3 11.7 ±1.3  1.7 ± 0.4 NK cells 3.0 ± 0.2 13.4 ± 1.2  4.5 ± 0.2

The ability of antibodies 28G11, 22F9, 20E6, 17G8, 24E3, and 2F8 to bindto human macrophage subsets was also examined. Briefly, CD14±cells wereobtained from StemExpress where they were isolated from whole blood of adonor via magnetic negative selection. Cells were cultured for 6 days inImmunocult macrophage medium (StemCell tech)+M-CSF (50 ng/ml) with thefollowing additions to skew the cells to specific macrophage subtypes(M1 macrophages: 50 ng/ml h-IFN-γ+10 ng/ml LPS; M2a macrophages: 10ng/ml h-IL4; M2b macrophages: immobilized IgG+100 ng/ml LPS; M2cmacrophages: 10 ng/ml IL10+20 ng/ml TGFβ). Cells were stained with CD14and Alexa 647-labeled anti-LAP antibodies 28G11_mIgG2a, 22F9_mIgG2a,20E6_mIgG2a, 17G8_hIgG1, 24E3_hIgG1, 2F8_(hyb), mIgG2a isotype control,or hIgG1 isotype control. Cells were gated as live, single cell prior toanalysis.

As shown in Table 10, the anti-LAP antibodies displayed very differentbinding patterns to isolated human macrophage subsets. Notably, 22F9bound a much higher percentage of all macrophage subpopulations than did28G11 or 20E6. 20E6 bound a higher percentage of M1 macrophages than did28G11.

TABLE 10 Binding to immune cells from healthy donors (expressed as %positive) Immune cell type 28G11 22F9 20E6 17G8 24E3 2F8 Isotype controlMacrophages 55.8% 85.0% 51.8% 24.8% 50.8% 40.0% 1.04% M1 macrophages15.2% 86.4% 46.1% 52.2% 50.1% 0.91% 2.34% M2a macrophages 55.6% 85.0%51.5% 20.6% 34.3% 29.1% 3.53% M2b macrophages 24.5% 90.8% 20.4% 50.7%66.7% 26.9% 2.14% M2c macrophages 29.1% 99.1% 28.2% 72.8% 84.5%  5.1%1.26%

In a separate experiment, the binding of the anti-LAP antibodies toactivated human CD4+ T cell populations was also assessed. CD4+ cellswere isolated from PBMCs using magnetic negative selection according toinstructions provided by the manufacturer (StemCell Tech). Cells wereactivated using a 1:1 ratio of Dynabeads (Thermo) to cells and culturedin advanced RPMI+10% FBS+30 U/ml human IL2 for 48 hours. Cells werestained with live/dead dye followed by CD4, CD25, and 28G11-IgG2a,22F9-IgG2a, 20E6-IgG2a, 17G8-hIgG1, 24E3-hIgG1, 2F8_(hyb), IgG1 isotypecontrol, or IgG2a isotype control for LAP expression. Cells were fixedand permeabilized for Foxp3 staining according to the manufacturerrecommendations (Ebiosciences) and stained for Foxp3. Cells were gatedas live, single cells, CD4+ and CD25+ prior to analysis. 22F9 was foundto bind a higher percentage of activated CD4+ cells than did the othertested anti-LAP antibodies.

Increased binding to specific cell populations is expected to beassociated with direct clinical benefit. The antibodies described hereinhibit TGFβ activation and release of the mature cytokine. Because TGFβacts in an autocrine or near-paracrine manner, selective binding tospecific cell populations will result in inhibition of the production ofmature TGFβ in the immediate proximity of the indicated cell population.Thus, for example, the increased binding of anti-LAP antibody 20E6 whencompared to antibody 28G11 to regulatory T cells would be expected toresult in selectively reduced TGFβ levels at the surface of those sameregulatory T cells. Given that TGFβ is a major driver of regulatory Tcell generation, this is expected to result in reduced numbers ofregulatory T cells in the tumor microenvironment and increased clinicalefficacy of 20E6 over 28G11. In a second example, the increased bindingof anti-LAP antibody 22F9 when compared to antibody 28G11 to macrophagesubsets would be expected to result in selectively reduced TGFβ levelsat the surface of those same macrophages. Given that TGFβ is a primarymechanism of cell-contact dependent macrophage inhibition of effector Tcell function, this is expected to result in reduced macrophage-mediatedinhibition and increased effector T cell function in the tumormicroenvironment and increased clinical efficacy of 20E6 over 28G11.

In some embodiments, the anti-LAP antibodies are of an isotype withactive effector function and enhanced binding of a specific anti-LAPantibody to a given cell population will result in increased depletionof that cell population by ADCC or CDC. Thus, for example, the increasedbinding of anti-LAP antibody 20E6 when compared to antibody 28G11 toregulatory T cells would be expected to result in increased ADCC orCDC-mediated depletion of those regulatory T cells in the tumormicroenvironment and increased clinical efficacy of 20E6 over 28G11. Ina second example, the increased binding of anti-LAP antibody 22F9 whencompared to antibody 28G11 to macrophage subsets would be expected toresult in increased ADCC- or CDC-mediated depletion of those macrophagesubsets in the tumor microenvironment and increased clinical efficacy of22F9 over 28G11.

The data presented in this example demonstrate that the finding that theanti-LAP antibodies described here bind differently to immune cellsubpopulations can be demonstrated in both murine and human systems andin both primary cell populations and transformed cell lines.

Example 8: Generation of Humanized Anti-LAP Antibodies

This Example describes the humanization of anti-LAP antibodies 28G11,22F9, and 20E6.

Models of 28G11, 22F9, and 26E10 (also referred to as 20E6) variableregions were built using Modeller, a program which uses multiplestructure templates to assemble a structural model. PDB code 3dv6 waschosen as the template for the 28G11 heavy chain (86% sequence identitywith the 28G11 VH domain) and 2zjs was chosen as the template for the28G11 light chain (97% sequence identity with 28G11 VL domain). PDB code1a6v was chosen as the template for the 22F9 heavy chain (89% sequenceidentity with the 22F9 V region), and PDB code 2xqy was chosen as thetemplate for the 22F9 light chain (94% identity with the 22F9 V region).PDB code 1a6v was chosen as the template for the 26E10 (20E6) heavychain (93% sequence identity with the 26E10 (20E6) V region), and PDBcode ljv5 was chosen as the template for the 26E10 (20E6) light chain(96% identity with the 26E10 V region). Structural models of thevariable domains were assembled and refined with Modeller.

To choose antibody acceptor framework sequences for the light and heavychains, an antibody sequence database and query tools were used toidentify suitable templates with the highest similarity to the murine28G11, 22F9, and 26E10 (20E6) sequences in canonical, interface, andVernier zone residues; the same length CDRs if possible (except CDR-H3);and a minimum required number of back mutations (i.e., changes offramework residue types from that of the human acceptor to that of themature murine antibody). Human germline sequences filled in with humanconsensus residues in the FR4 framework region were considered as well.

Based on the above analysis, CDR sequences of 28G11 were grafted ontoIGHV3-72*01(H0) and IGKV1-27*01(L0) germlines. For humanization of 22F9,CDR sequences of 22F9 were grafted onto IGHV1-46*01 (H0) andIGKV1-39*01(L0) germlines. For humanization of 20E6, CDR sequences of20E6 were grafted onto human IGHV1-2*05(H0) and IGKV1-33*01(L0)germlines. Back substitutions to mouse sequences in the heavy and/orlight chains of these humanized antibodies were introduced based onanalysis of homology models. Substitutions were also introduced (asdiscussed in the Examples that follow) to remove potential deamidationand isomerization sites. Each of these candidates were tested forvarious functions, including binding to cells overexpressing LAP-TGFβ1,as described in the Examples below.

Example 9: Characterization of Humanized 28G11 Candidates and LiabilitySite-Removed 28G11 Variants

In this Example, the characteristics of various humanized 28G11candidates were examined to identify humanized antibodies that retainedthe function of the parental antibodies (e.g., binding to TGFβ1). Alsoexamined were deamidation/isomerization site-removed 28G11 variants.Table 11 summarizes the various humanized 28G11 constructs (heavy andlight chain sequences) used in this Example. The sequences of theseconstructs are provided in Table 34.

TABLE 11 Exemplary 28G11 antibodies and antigen binding fragments SEQ IDName Description 32 28G11_H0 Humanized 28G11 heavy chain sequence withCDR1, CDR2 and CDR3 sequences from parental murine antibody insertedinto the human IGHV3- 72*01 germline with a consensus framework 4sequence 34 28G11_H1 Humanized 28G11_H0 heavy chain sequence whichincludes amino substitutions S30T and V48L 36 28G11_H2 Humanized28G11_H0 heavy chain sequence which includes amino substitutions S30T,V48L, D73N, K75Q, 04N76S, and V109L 39 28G11_H2.1 Humanized 28G11_H2heavy chain sequence which includes amino substitution N56Q 41 28G11_H2aHumanized 28G11_H0 heavy chain sequence which includes aminosubstitutions S30T and D73N 43 28G11_H2b Humanized 28G11_H0 heavy chainsequence which includes amino substitutions S30T, N56Q and D73N 4528G11_H2b_ H2b heavy chain with a variant human IgG4 hIgG4mut constantregion; the variant human IgG4 constant region has the sequence of SEQID NO: 197. 217 28G11_L0 Humanized 28G11 light chain sequence with CDR1,CDR2 and CDR3 sequences from parental murine antibody inserted into thehuman IGKV1- 27*01 germline with a huIGKJ2 framework 4 sequence 4728G11_L1 Humanized 28G11_L0 light chain sequence which includes aminosubstitutions F71Y and Y87F 49 28G11_L2 Humanized 28G11_L0 light chainsequence which includes amino substitutions V43T, P44V, F71Y, and Y87F51 28G11_L3 Humanized 28G11_L0 light chain sequence which includes aminosubstitutions V19L, T22S, V43T, P44V, F71Y, and Y87F 53 28G11_L3aHumanized 28G11_L0 light chain sequence which includes aminosubstitutions V19L, T22S, P44V, and F71Y.

In the first experiment, the various humanized 28G11 candidates weretested for retention of binding to human LAP-TGFβ1 by competition withbiotin-28G11 in a flow cytometry assay.

P3U1 cells over-expressing human GARP and LAP-TGFβ1 were placed in around-bottom 96-well plate at density of 40,000 cells/well. The cellswere washed with FACS buffer (25 mM HEPES, 2 mM EDTA, 2% fetal bovineserum in Hank's Balanced Salt Solution). 50 μL of 1:200 diluted TruStainMouse FcX (BioLegend) was added to each well, followed by 50 μL of 100ng/μL of humanized 28G11 construct (H0L0, H0L1, H0L2, H0L3, HILO, H1L1,H1L2, H1L3, H2L0, H2L1, H2L2, H2L3). See Table 34. The antibody wasallowed to bind for 10 minutes. 50 μL of 6.2 ng/μL of biotinylatedmurine hybridoma 28G11_(hyb) was added to each well. The biotinylatedantibody was allowed to bind for 10 minutes, and then the cells werewashed twice with FACS buffer. The cells were labeled for 15 minuteswith allophytocyanin-streptavidin (BioLegend), washed twice with FACSbuffer, and analyzed by flow cytometry.

As shown in FIG. 12, all constructs with H0, H1, or L0 failed to blockbiotin-28G11 binding to human LAP-TGFβ1, indicating that these humanizedframeworks do not allow the CDR loops to adopt the correct structure fortight LAP-TGFβ1 binding. 28G11_H2L1, 28G11_H2L2, and 28G11_H2L3 didcompete with the parental murine antibody. These constructs werebiotinylated to confirm specific binding.

Next, direct binding of the humanized 28G11 variants to P3U1 cellsover-expressing human GARP and LAP-TGFβ1 was tested. Briefly, P3U1 cellsoverexpressing human GARP and LAP-TGFβ1 were plated, washed andFcX-treated as described above. The cells were incubated with 10 μg/mLbiotinylated 28G11_H0L3, 28G11_H2L0, 28G11_H2L1, 28G11_H2L2 or28G11_H2L3 for 20 minutes at 4° C. The cells were washed, stained withAPC-streptavidin, washed again and analyzed by flow cytometry asdescribed above. Consistent with the competition experiment, 28G11_H0L3and 28G11_H2L0 did not bind, but 28G11_H2L1, 28G11_H2L2 and 28G11_H2L3showed potent binding to human LAP-TGFβ1 (FIG. 13).

The ability of 28G11_H2L1, 28G11_H2L2, and 28G11_H2L3, which showed themost potent binding among the humanized 28G11 variants tested in thestudies described above, to inhibit TGFβ1 activation was tested in anELISA-based assay. Briefly, P3U1 cells expressing human TGFβ werecultured overnight in serum free advanced medium in round bottom tissueculture plates and treated the following day with the humanized 28G11variants (2-fold serial dilutions starting with 20 ug/ml) for 24 hours.Active TGFβ1 was detected in the supernatant of cell cultures byutilizing a commercially available human TGFβ1 ELISA kit (R&D Systems)according to the manufacturer instructions. As shown in FIGS. 14A-14D,28G11_(hyb), 28G11_H2L1, and 28G11_H2L3, but not 28G11_H2L2, inhibitedTGFβ1 activation.

Based on the studies described above, the best binding was observed with28G11_H2L3. As shown below, both the H2 heavy chain and L3 light chaininclude 6 positions that are back-mutated to murine residues from thehuman framework (back-mutated residues are in lower case letters andunderlined.

28G11_H2: (SEQ ID NO: 35) EVQLVESGGGLVQPGGSLRLSCAAS GFTFtDYYMSWVRQAPGKGLEWlG FIRNKPNGYTTEYSASVKGRFTISRDnSqsSLYLQMNSLKTEDTAVYYCAR YTGGGYFDY WGQGTLlTVSS 28G11_L3: (SEQ ID NO: 50)DIQMTQSPSSLSASVGDRlTIsC RASQDISNYLN WYQQKPGKtvKLLIY YTSRLHSGVPSRFSGSGSGTDyTLTISSLQPEDVATYfC QQGDTLPWT FGQGTKLEIK

28G11_H2L3 was used as the basis for adding back individualsubstitutions to determine which of the back-mutated positions couldtolerate human residues. FIGS. 15A and 15B show the effects that variouscombinations of reversions back to human residues had on binding toHT1080 cells overexpressing human LAP-TGFβ1. For example, thecombination of HC_N73D+LC_Y71F eliminated binding, and the combinationof HC_T30S+LC_V44P had reduced binding. The LC_L19V construct was poorlyexpressed. From the data in FIG. 15A, it was concluded that the L48V,Q75K, S76N, and L109V substitutions could be tolerated on the heavychain (resulting in sequence 28G11_H2a) and the T43 V and F87Y could betolerated on the light chain (resulting in sequence 28G11_L3a).28G11_H2aL3a was tested for binding to HT1080 cells over-expressinghuman LAP-TGFβ1. See Table 12 and Table 34. As shown in FIG. 15B, thisconstruct binds as well as a mouse-human chimera containing the originalvariable region domains of the parental murine 28G11 antibody.

The 28G11_H2L3 sequence contains a ‘NG’ dipeptide in the CDR2 regionthat can undergo a deamidation reaction to produce aspartate oriso-aspartate at the position of the asparagine residue. To preventthis, a N56Q substitution was introduced into the sequence(28G11_H2.1L3). This antibody binds HT1080-huB1 cells slightly betterthan the original sequence (FIG. 15B).

Example 10: Characterization of Humanized 22F9 Candidates and LiabilitySite-Removed 22F9 Variants

In this Example, the characteristics of various humanized 22F9candidates were examined to identify humanized antibodies that retainedthe function of the parental antibodies (e.g., binding to TGFβ1). Alsoexamined were deamidation/isomerization site-removed 22F9 variants.Table 12 summarizes the various 22F9 variants used in this Example. Thesequences of these constructs are provided in Table 34.

TABLE 12 Exemplary 22F9 antibodies and antigen binding fragments SEQ IDName Description 72 22F9_H0 Humanized 22F9 heavy chain sequence whichthe CDR1, CDR2 and CDR3 sequences of the parental murine antibody areinserted into the IGHV1-46*01 germline with a consensus framework 4sequence 74 22F9_H0.1 Humanized 22F9_H0 heavy chain sequence whichincludes amino substitutions S7P, K12V, V20L, R38K, A40R, N54Q, R66K,V67A, M69L, T73K, T755, V78A, and D102A 76 22F9_H1 Humanized 22F9_H0heavy chain sequence which includes amino substitutions M48I, R71V, andR94Y 78 22F9_H1.1 Humanized 22F9_H0 heavy chain sequence which includesamino substitutions S7P, K12V, V20L, R38K, A40R, M48I, N54Q, R66K, V67A,R71V, T73K, T755, R94Y, and D102A 81 22F9_H2 Humanized 22F9_H0 heavychain sequence which includes amino substitutions M48I, M69L, R71V,V78A, and R94Y 83 22F9_H2.1 Humanized 22F9_H0 heavy chain sequence whichincludes amino substitutions S7P, K12V, V20L, R38K, A40R, M48I, N54Q,R66K, V67A, M69L, R71V, T73K, V78A, R94Y, and D102A 86 22F9_H3 Humanized22F9_H0 heavy chain sequence which includes amino substitutions M48I,M69L, R71V, T73K, T755, V78A, and R94Y 88 22F9_H3.1 Humanized 22F9_H0heavy chain sequence which includes amino substitutions S7P, K12V, V20L,M48I, N54Q, M69L, R71V, T73K, T755, V78A, R94Y, and D102A 91 22F9_H4Humanized 22F9_H0 heavy chain sequence which includes aminosubstitutions R38K, A40R, M48I, R66K, V67A, M69L, R71V, T73K, T755,V78A, and R94Y 93 22F9_H5 Humanized 22F9_H0 heavy chain sequence whichincludes amino substitutions S7P, K12V, V20L, R38K, A40R, M48I, R66K,V67A, M69L, R71V, T73K, T75S, V78A, and R94Y 96 22F9_H5.2 Humanized22F9_H0 heavy chain sequence which includes amino substitutions S7P,K12V, V20L, R38K, A40R, M48I, N54Q, R66K, V67A, M69L, R71V, T73K, T75S,V78A, R94Y, and D102A 99 22F9_H7 Humanized 22F9_H0 heavy chain sequencewhich includes amino substitutions S7P, K12V, V20L, M48I, N54Q, R71V,T73K, R94Y, and D102A 100 22F9_H7a_ 22F9_H7 heavy chain with a varianthuman IgG4 hIgG4mut constant region; the variant human IgG4 constantregion has the sequence of SEQ ID NO: 197. 102 22F9_H7a Humanized22F9_H0 heavy chain sequence which includes amino substitutions Q1E,S7P, K12V, V20L, M48I, N54Q, R71V, T73K, R94Y, and D702A 103 22F9_H7a_22F9_H7a heavy chain with a variant human IgG4 hIgG4mut constant region;the variant human IgG4 constant region has the sequence of SEQ ID NO:197. 105 22F9_L0 Humanized 22F9 light chain sequence which the CDR1,CDR2 and CDR3 sequences of the parental murine antibody are insertedinto the IGKV1-39x01 germline with a IGKJ4 framework 4 sequence 10722F9_L1 Humanized 22F9_L0 light chain sequence which includes amino acidsubstitutions A43P and D70H 109 22F9_L2 Humanized 22F9_L0 light chainsequence which includes amino acid substitutions A13V, V19A, A43P, D70H,L78V, and V104L

First, the ability of 22F9H0L0, HILO, H2L0, H3L0, H4L0, H5L0, H0L1,H1L1, H2L1, H3L1, H4L1, H5L1, H0L2, H1L2, H2L2, H3L3, H4L2, and H5L2 tobind to HT1080 cells overexpressing human TGFβ1 was tested by flowcytometry. HT1080 cells over-expressing human LAP-TGFβ1 were placed in around-bottom 96-well plate at density of 40,000 cells/well. The cellswere washed with FACS buffer (25 mM HEPES, 2 mM EDTA, 2% fetal bovineserum in Hank's Balanced Salt Solution). 50 μL of 1:200 diluted TruStainHuman FcX (BioLegend) was added to each well, followed by 50 μL of 100ng/μL of humanized 22F9 construct. The antibody was allowed to bind for20 minutes. The cells were washed twice with FACS buffer. The cells werelabeled for 15 minutes with Alexa647-anti Human IgG (JacksonImmunoresearch), washed twice with FACS buffer, and analyzed by flowcytometry. As shown in FIG. 16, 22F9_H2L0, H3L0, H4L0, H5L0, H2L1, H3L1,H4L1, H5L1, and H4L2 showed some binding relative to control HT1080cells and 22F9H0L0. The strongest binding was observed with antibodiescontaining the H5 heavy-chain sequence.

Binding to human TGFβ1 of a subset of candidates was also tested bybio-layer interferometry (Octet). A streptavidin coated tip wasequilibrated for 60 seconds in binding buffer (10 mM sodium phosphate,150 mM sodium chloride, 1% bovine serum albumin, 0.05% sodium azide).The tip was then dipped in 10 μg/mL biotinylated antibody in bindingbuffer. After 15 seconds, the tip was washed in binding buffer for 60seconds, and then dipped in 0-24 nM Fc-human LAP. Analyte associationwas measured for 5 minutes. The tip was transferred to binding bufferalone, and analyte dissociation was measured for 5 minutes. Theassociation and dissociation data were fit to a 1:1 binding model. Asshown in Table 13, of the candidates tested, 22F9_H5L0 showed thehighest signal change on binding, and 22F9_H4L0 showed the tightestaffinity.

TABLE 13 Binding data for 22F9 antibodies and antigen binding fragmentsk_(on) k_(off) K_(D) Loading Binding Antibody (×10⁵ M⁻¹s⁻¹) (×10⁻³ s⁻¹)(nM) (nm) (nm) 22F9_hIgG1 4.50 1.68 3.74 1.13 0.27 22F9_H3L0 7.78 2.142.75 0.99 0.023 22F9_H4L0 8.10 1.32 1.63 0.90 0.057 22F9_H4L2 5.98 1.372.30 1.17 0.07 22F9_H5L0 5.10 1.7 3.35 1.22 0.13 22F9_H5L1 5.19 1.222.34 1.29 0.08 22F9_H5L2 6.29 2.46 3.91 1.25 0.06

In another experiment, size exclusion chromatography (SEC) was used toassess aggregation of the candidates. Each protein was diluted to 1mg/mL in phosphate buffered saline, pH 7.4. A Sepax SEC-300 column wasequilibrated with 10 mM sodium phosphate, 150 mM sodium chloride, 0.05%(v/v) sodium azide, pH 7.4. 10 μL of 1 mg/mL antibody was injected ontothe column. Eluted proteins were detected by absorbance at 280 nm. TheIgG monomer peak was identified by comparing its retention time againsta set of gel-filtration molecular weight standards (Bio-Rad). Theresults are shown in Table 14.

TABLE 14 22F9 chromatographic data Antibody RT (min) Area (mAU * s) %monomer 22F9_mIgG2a 7.956 341 >99% 22F9_hIgG1 8.035 259 >99% 22F9_H0L07.93 160  77% 22F9_H4L0 8.083 168 >99% 22F9_H4L1 8.178 180 >99%22F9_H4L2 8.161 214 >99% 22F9_H5L0 8.02 220 >99% 22F9_H5L1 8.106219 >99% 22F9_H5L2 8.11 206 >99%

Most of the tested candidates showed little aggregation, as reflected inthe high percentage of monomers (>99%). Candidates with lower thanexpected Octet binding signals also had smaller SEC peaks. Thesignificantly lower total area for 22F9_H4L0 implies the aggregates aretoo large to enter the column, whereas all 22F9_H5 constructs were wellbehaved. FIG. 17A shows size exclusion-high performance liquidchromatography (SE-HPLC) results for 22F9_hIgG1, 22F9_H0L0, and22F9_H4L0, and FIG. 17B shows the results for 22F9_H5L0, H5L1, and H5L2.

The H5L0 candidate, the sequence of which is provided below, was furthercharacterized by reverting murine residues back to the correspondinghuman residue by single-site substitution to determine which murineresidues are essential. Additional substitutions were made to removepotential deamidation sites (N54S, N54H, N54A, N54Q) and isomerizationsites (D102E, D102A, D102G).

22F9_H5: (SEQ ID NO: 92) QVQLVQpGAEVvKPGASVKlSCKAS GYTFTSYWMHWVkQrPGQGLEWiG MIHP

SGSTNYNEKFKS kaTlTvDkSsSTaYMELSSLRSEDTAVYYCAy YDY

GFFDV WGQGTLVTVSS

As shown in FIG. 18A, all single-site substitutions reverting murineframework residues back to human substantially reduced binding toLAP-TGFβ1. As shown in FIG. 18B, the N54Q and D102A substitutionsretained the most binding activity of all substitutions at thosepositions, and a double N54Q/D102A variant (22F9_H5.2) had the strongestbinding signal of all antibodies tested in this assay. See also Table 12and Table 34.

As the single variants did not yield any reduction in the number ofmurine residues, an alternative strategy was employed to make amino-acidsubstitutions in groups. These constructs, labeled 22F9_H0.1, 22F9_H1.1,22F9_H2.1, and 22F9_H3.1, were assayed for binding to HT1080 cellsover-expressing human LAP-TGFβ1 as described above. As shown in FIG.18C, the 22F9_H0.1 variant did not bind human LAP-TGFβ1, while the22F9_H1.1, 22F9_H2.1 and 22F9_H3.1 variants did bind human LAP-TGFβ1.

Example 11: Characterization of Humanized 20E6 Candidates and LiabilitySite-Removed 20E6 Variants

In this Example, the characteristics of various humanized 20E6candidates were examined to identify humanized antibodies that retainedthe function of the parental antibodies (e.g., binding to TGFβ1). Alsoexamined were deamidation/isomerization site-removed 20E6 variants.Table 15 summarizes the various 20E6 variants used in this Example. Thesequences of these constructs are provided in Table 34. Notably, L1includes 3 murine back-substitutions, i.e., P44V (which is at the VH/VLinterface and could potentially affect domain pairing and stability),F71Y (a canonical residue which could potentially affect the structureof CDR L2), and Y87F (which is at the VH/VL interface and couldpotentially affect domain pairing and stability).

TABLE 15 Exemplary 20E6 antibodies and antigen binding fragments SEQ IDName Description 128 20E6_H0 Humanized 20E6 heavy chain sequence whichthe CDR1, CDR2 (extended definition) and CDR3 sequences of the parentalmurine antibody are inserted into the IGHV1-2*02 germline with aconsensus framework 4 sequence 131 20E6_H0.1 Humanized 20E6 heavy chainsequence which the CDR1, CDR2 and CDR3 sequences of the parental murineantibody are inserted into the IGHV1-2*02 germline with a consensusframework 4 sequence 134 20E6_H0.2 Humanized 20E6_H0.1 heavy chainsequence which includes the amino acid substitution N54Q 219 20E6_H0.2aHumanized 20E6_H0.1 heavy chain sequence which includes the amino acidsubstitutions Q1E and N54Q 136 20E6_H0.3 Humanized 20E6_H0.1 heavy chainsequence which includes the amino acid substitution N54G 137 20E6_H0.4Humanized 20E6_H0.1 heavy chain sequence which includes the amino acidsubstitution N54A 138 20E6_H0.5 Humanized 20E6_H0.1 heavy chain sequencewhich includes the amino acid substitution N54S 139 20E6_H0.6 Humanized20E6_H0.1 heavy chain sequence which includes the amino acidsubstitution N54H 140 20E6_H0.7 Humanized 20E6_H0.1 heavy chain sequencewhich includes the amino acid substitution N54L 141 20E6_H0.8 Humanized20E6_H0.1 heavy chain sequence which includes the amino acidsubstitution N54D 135 20E6_H0.2 hIgG4 20E6_H0.2 heavy chain with avariant human IgG4 mut constant region; the variant human IgG4 constantregion has the sequence of SEQ ID NO: 197. 220 20E6_H0.2a hIgG420E6_H0.2a heavy chain with a variant human IgG4 mut constant region;the variant human IgG4 constant region has the sequence of SEQ ID NO:197. 145 20E6_H1 Humanized 20E6_H0 heavy chain sequence which includesamino acid substitutions M48I, M69L, R71V 147 20E6_H2 Humanized 20E6_H0heavy chain sequence which includes amino acid substitutions M48I, M69L,R71V, T73K, T75S 149 20E6_H3 Humanized 20E6_H0 heavy chain sequencewhich includes amino acid substitutions R38K, A40R, M48I, R66K, V67A,M69L, R71V, T73K, T75S 151 20E6_H4 Humanized 20E6_H0 heavy chainsequence which includes amino acid substitutions S7P, K12V, V20L, R38K,A40R, M48I, R66K, V67A, M69L, R71V, T73K, T75S 153 20E6_L0 Humanized20E6 light chain sequence which the CDR1, CDR2 and CDR3 sequences of theparental murine antibody are inserted into the IGKV1-39*01 germline witha IGKJ2 framework 4 sequence 155 20E6_L1 Humanized 20E6_L0 light chainsequence which includes amino acid substitutions P44V, F71Y, Y87F 15720E6_L0_P44V 20E6_L0 having a P to V mutation at position 44 15920E6_L0_F71Y 20E6_L0 having a F to Y mutation at position 71 16120E6_L0_Y87F 20E6_L0 having a Y to F mutation at position 87

First, the ability of the humanized 20E6 candidates to bind to HT1080cells overexpressing human TGFβ1 was tested by flow cytometry. HT1080cells over-expressing human LAP-TGFβ1 were placed in a round-bottom96-well plate at density of 40,000 cells/well. The cells were washedwith FACS buffer (25 mM HEPES, 2 mM EDTA, 2% fetal bovine serum inHank's Balanced Salt Solution). A volume (50 μL) of 1:200 dilutedTruStain Human FcX (BioLegend) was added to each well, followed by 50 μLof 100 ng/μL of humanized 20E6 antibody construct. The antibody wasallowed to bind for 20 minutes. The cells were washed twice with FACSbuffer. The cells were labeled for 15 minutes with Alexa647-anti HumanIgG (Jackson Immunoresearch), washed twice with FACS buffer, andanalyzed by flow cytometry. As shown in FIGS. 19A and 19B, candidateswhich include the L1 light chain showed greater binding to HT1080-huβ1cells than to control HT1080 cells.

The binding of humanized 20E6 candidates to human LAP-TGFβ1 was alsotested by bio-layer interferometry (Octet). As shown in Table 16,20E6_H(0-4)L1 had comparable binding kinetics, consistent with the flowcytometry results.

TABLE 16 Octet binding data for 20E6 Antibody k_(on) (×10⁵ M⁻¹s⁻¹)k_(off) (×10⁻³ s⁻¹) K_(D) (nM) 20E6_hIgG1 0.98 9.97 1.02 20E6_H0L1 1.598.14 0.51 20E6_H1L1 1.41 6.77 0.48 20E6_H2L1 1.57 7.47 0.48 20E6_H3L11.48 7.02 0.47 20E6_H4L1 1.56 7.29 0.47

As discussed above, the L1 light chain has 3 murine back-substitutions,i.e., P44V, F71Y, and Y87F. To determine whether all three of thesemurine back-substitutions are necessary for binding to LAP-TGFβ1, eachsubstitution was introduced individually into the L0 light chain andcompared to 20E6 H0L1. As shown in FIG. 20A, all three murineback-substitutions are necessary for binding to HT1080 cellsover-expressing LAP-TGFβ1.

The CDR grafting used to generate the 20E6 H0 sequence used an extendeddefinition of the heavy-chain CDR2, which resulted in the incorporationof additional murine residues in 20E6 H0. To reduce the risk ofimmunogenicity, a variant of 20E6 H0 was made with the traditional(Kabat) definition for heavy-chain CDR2. This construct, 20E6 H0.1,bound to human LAP-TGFβ1, as well as 20E6 H0, indicating that thesemurine residues are not needed (FIG. 20B).

Example 12: Binding of Anti-LAP Antibodies to Human LAP-TGFβ1, 2 and 3

This Example describes testing the specificity of humanized 20E6(20E6_H0.2aL1_IgG1) antibody, which has the heavy and light chainsequences of SEQ ID NOs: 219 and 155, respectively) for binding to humanLAP-TGFβ isoforms 1, 2, and 3 using bio-layer interferometry.

The 20E6_H0.2aL1_IgG1 antibody was biotinylated using EZ-LinkSulfoNHS-LC-Biotin (ThermoFisher). A streptavidin-functionalized tip wasequilibrated in binding buffer (10 mM sodium phosphate, 150 mM sodiumchloride, 1% (w/v) bovine serum albumin, 0.05% (w/v) sodium azide, pH7.4) and dipped in a 10 μg/mL solution of biotinylated anti-LAP inbinding buffer for 30 seconds to load the tip with antibody. Theantibody-loaded tip was then washed in binding buffer and placed in asolution containing 0-24 nM of a fusion protein having a human IgG1 Fcdomain fused to either human LAP-TGFβ1, human LAP-TGFβ2 or humanLAP-TGFβ3. The antigen was allowed to bind to antibody for 5 minutes(association phase), and then the tip was moved to binding buffer(dissociation phase). The association and dissociation phases were fitto a 2:1 heterogenous ligand binding model to determine the binding rateconstants. As shown in FIG. 21A and Table 17, 20E6_H0.2aL1_IgG1 boundwith sub-nanomolar affinity to human LAP-TGFβ1, but not to humanLAP-TGFβ2 (FIG. 21B) or LAP-TGFβ3 (FIG. 21C). These data demonstratedthat the 20E6_H0.2aL1_IgG1 antibody specifically binds to humanLAP-TGFβ1 and there was no binding to LAP-TGFβ2 and LAP-TGFβ3.

TABLE 17 Binding of anti-LAP antibody 20E6_H0.2aL1_IgG1 to humanLAP-TGFβ1, 2 and 3 Sample k_(on)(×10⁶ k_(off) K_(D) k_(on,2) (×10⁶k_(off,2) K_(D,2) ID M⁻¹s⁻¹) (×10⁻⁴ s⁻¹) (nM) M⁻¹s⁻¹) (×10⁻⁴ s⁻¹) (nM)Fc-huB1 3.24 369 11.4 1.18 4.09 0.35 Fc-huB2 — — — — — — Fc-huB3 — — — —— —

Example 13: Binding of Anti-LAP Antibodies to Mouse, Rat, CynomolgusMonkey, and Human LAP-TGFβ1

This Example describes the testing of species specificity of20E6_H0.2aL1_hIgG1 antibody and 20E6_H0.2aL1_hIgG4mut antibody (whichhas the heavy and light chain sequences of SEQ ID NOs: 220 and 155,respectively) to bind to LAP-TGFβ1 from several species using bio-layerinterferometry.

The binding of 20E6_H0.2aL1_IgG1 antibody and 20E6_H0.2aL1_hIgG4mutantibody to LAP-TGFβ1 of various species was examined by bio-layerinterferometry using the method described in Example 12, except that theantibody-loaded tip was placed in a solution containing 0-24 nM of afusion protein having a human IgG1 Fc domain fused to either mouse, rat,cynomolgus monkey, or human LAP-TGFβ1. As shown in Tables 18 and 19,respectively, 20E6_H0.2aL1_hIgG1 antibody and 20E6_H0.2aL1_hIgG4mutantibody bound with nanomolar affinity to LAP-TGFβ1 from all speciestested.

TABLE 18 Binding data of 20E6_H0.2aL1_IgG1 antibody to LAP-TGFβ1 ofvarious species LAP-TGFβ1 K_(D) (nM) k_(on) (×10⁶ M⁻¹ s⁻¹) k_(off)(×10⁻⁴ s⁻¹) K_(D,2) (nM) k_(on,2) (×10⁶ M⁻¹ s⁻¹) k_(off,2) (×10⁻⁴ s⁻¹)Human 16.51 ± 0.01^(a) 2.15 ± 0.18 355 ± 14 0.97 ± 0.10 0.68 ± 0.02 6.57± 0.67 Cyno 15.85 ± 0.01  2.49 ± 0.30 394 ± 22 0.71 ± 0.13 1.03 ± 0.067.28 ± 1.24 Rat 13.38 ± 0.01  3.30 ± 0.25 441 ± 16 0.68 ± 0.07 1.14 ±0.05 7.68 ± 0.77 Mouse 14.19 ± 0.01  2.42 ± 0.26 343 ± 19 0.56 ± 0.140.70 ± 0.04 3.90 ± 0.98 ^(a)All values reported as average ± standarddeviation from triplicate measurements

TABLE 19 Binding data of 20E6_H0.2aL1_hIgG4mut antibody to LAP-TGFβ1 ofvarious speciesLAP-TGFβ1 K_(D) (nM) k_(on) (×10⁶ M⁻¹ s⁻¹) k_(off) (×10⁻⁴s⁻¹) K_(D,2) (nM) k_(on,2) (×10⁶ M⁻¹ s⁻¹) k_(off,2) (×10⁻⁴ s⁻¹) Human10.72 ± 0.01 2.8 ± 0.3 303 ± 16 0.51 ± 0.12 0.70 ± 0.03  3.6 ± 0.8 Cyno26.14 ± 0.05 2.0 ± 0.4 524 ± 36 1.53 ± 0.18 0.94 ± 0.06 14.3 ± 1.4 Rat11.14 ± 0.01 3.6 ± 0.4 406 ± 20 0.51 ± 0.06 1.70 ± 0.08  8.7 ± 0.9 Mouse16.88 ± 0.02 2.8 ± 0.4 478 ± 27 1.31 ± 0.11 0.84 ± 0.04 11.0 ± 0.8^(a)All values reported as average ± standard deviation from triplicatemeasurements

Example 14: Alanine Scanning Mutagenesis of CDRs in Humanized 20E6

This Example describes the testing of binding of human LAP-TGFβ1 tovariants of 20E6_H0.2aL1_IgG1 antibody using bio-layer interferometry.

A total of 49 single alanine substitutions were made in the heavy andlight chain CDRs of the antibody to identify critical residues for humanLAP-TGFβ1 binding. In addition, residue A25 in the light chain wassubstituted with glycine. Plasmid DNA for these variants was transfectedinto ExpiCHO cells in 1 mL cultures on 24-well plates, along with wellscontaining no DNA (negative control) and wells transfected with DNA forthe wild-type antibody (positive control). IgG concentrations in eachwell were determined by binding to Protein A-functionalized tips. Tipswere dipped into wells containing the supernatants from each culture.IgG concentrations were determined by comparing the rate of signalchange for each well against the rate of signal change for a standardcurve of purified 20E6_H0.2aL1_hIgG1 antibody diluted into ExpiCHOmedia. CHO supernatants were diluted to 0.6 μg/mL in media. Astreptavidin-functionalized tip was equilibrated in binding buffer (10mM sodium phosphate, 150 mM sodium chloride, 1% (w/v) bovine serumalbumin, 0.05% (w/v) sodium azide, pH 7.4) and then dipped in a 5 μg/mLsolution of biotinylated human LAP-TGFβ1 in binding buffer for 60seconds to load the tip with antigen. The antigen-loaded tip was thenwashed in binding buffer and placed in the diluted CHO supernatant. Theantigen was allowed to bind to antibody for 5 minutes (associationphase), and then the tip was moved to binding buffer (dissociationphase). The association and dissociation phases were fit to a 1:1binding model to determine binding rate constants.

As shown in FIGS. 22A-22D and Table 20, alanine substitutions atpositions 50, 99, 101, 102, 103, 104, 105 in the heavy chain, andpositions 24, 28, 29, 32, 50, 53, 89, 90, 91, 92, 94, 95, 96 and 97 inthe light chain had modest to severe impacts on binding affinity,indicating that these residues are involved in binding to humanLAP-TGFβ1.

Example 15: Mono-Vs. Bi-Valent Binding of Anti-LAP Antibodies to HumanLAP-TGFβ1

This Example compared the monovalent binding and bivalent binding of20E6_H0.2aL1_hIgG1 antibody to human LAP-TGFβ1.

F(ab′)2 fragments of 20E6_H0.2aL1_hIgG1 antibody were generated with theFragIT kit (Genovis) following the manufacturer's instructions. TheF(ab′)₂ was then treated with 10 mM 2-Mercaptoethylamine-HCl (2-MEA) togenerate Fab′ fragments. To test binding to cells over-expressing humanLAP-TGFβ1, 4×10⁵ each of (a) P3U1 cells and (b) P3U1 cellsoverexpressing human GARP and human LAP-TGFβ1 were cultured in 96-wellplates. The plates were centrifuged for 5 min at 1,500 rpm, liquid wasremoved, and cells were resuspended with 200 μL FACS buffer. The plateswere centrifuged again, diluted primary antibody was added to each well,and the plates were incubated on ice for 20 minutes, followed bycentrifugation. The cells were resuspended in 200 μL FACS buffer,centrifuged again, and resuspended in 50 μL diluted secondary antibody(Alexa647-anti-Human IgG). The plates were incubated on ice for 20minutes in the dark, washed twice with 200 μL FACS buffer, and cellsfrom each well (in 200 μL FACS buffer) were read on the Attune NXTinstrument. As shown in FIGS. 23A and 23B, all three constructs boundthe P3U1 cells over-expressing GARP and LAP-TGFβ1.

Example 16: Binding of Anti-LAP Antibodies to Human LAP-TGFβ1 in thePresence of Anchor Proteins

This Example describes the testing of the binding of 20E6_H0.2aL1_hIgG1antibody to human LAP-TGFβ1 in the presence of anchor proteins.

LAP-TGFβ is anchored to the extracellular matrix through LTBP, and tothe surface of immunosuppressive cells via GARP or LRRC33. Soluble formsof human LTBP1 and GARP were prepared to assess the influence of theanchor protein on anti-LAP antibody binding. The ECR3E fragment(described in Annes et al. JCB 2004; 165:723) consists of the thirdcysteine-rich domain of human LTBP1, flanked by EGFR-like domains. Thisconstruct contains all of the elements necessary for covalent attachmentto LAP-TGFβ, forming a soluble complex. A soluble GARP-LAP-TGFβ complexwas prepared by co-expressing human LAP-TGFβ1 with a chimera comprisedof the extracellular domain of human GARP with the transmembrane andcytosolic domains of meprinα (described in Fridrich et al. PLoS ONE.2016; 11 (4): e0153290).

A streptavidin-functionalized tip was equilibrated in binding buffer (10mM sodium phosphate, 150 mM sodium chloride, 1% (w/v) bovine serumalbumin, 0.05% (w/v) sodium azide, pH 7.4) and dipped in a 5 μg/mLsolution of biotinylated antibody (i.e., 20E6_H0.2aL1_hIgG1, murine16F4, or MHG8, a GARP-specific murine IgG2a described in Lienart et al.Science 2018; 362:952-956) in binding buffer for 30 seconds to load thetip with antibody. The antibody-loaded tip was then washed in bindingbuffer and placed in a solution containing 0-24 nM of a fusion proteincontaining a human IgG1 Fc domain fused to either human LAP-TGFβ1, thesoluble GARP-LAP-TGFβ1 complex, or the soluble ECR3E-LAP-TGFβ1 complex.The antigen was allowed to bind to antibody for 5 minutes (associationphase), and then the tip was moved to binding buffer (dissociationphase). The binding and dissociation data for 16F4 and MHG8 werewell-fit by a 1:1 binding model. The association and dissociation phasesfor 20E6_H0.2aL1_hIgG1 antibody were fit to a 2:1 heterogenous ligandbinding model to determine the binding rate constants.

As shown in FIG. 24 and Table 21, the 20E6_H0.2aL1_hIgG1 antibody boundto free LAP-TGFβ1 and the soluble GARP-LAP-TGFβ1 complex with nanomolaraffinity, but not to the ECR3E-LAP-TGFβ1 complex. As expected, theanti-GARP antibody (MHG8) bound to the GARP-LAP-TGFβ1 complex, but notto free LAP-TGFβ1 or the ECR3E-LAP-TGFβ1 complex. 16F4 bound tightly toall three constructs.

TABLE 20 Human LAP-TGFb1 binding effects of CDR substitutions in20E6_H0.2aL1_hIgG1 Variant K_(D,WT)/K_(D,Ala) WT #4 1.00 VH_G26A 1.40VH_Y27A 1.29 VH_T28A 0.97 VH_F29A 1.16 VH_T30A 0.94 WT #1 0.90 VH_S31A0.60 VH_Y32A 1.00 VH_W33A 0.12 VH_M34A 0.84 VH_H35A 2.07 VH_R50A 0.00VH_I51A 0.73 VH_D52A 0.71 VH_P53A 0.87 VH_Q54A 0.67 VH_S55A 0.96 VH_G56A1.62 VH_G57A 0.37 VH_I58A 0.52 VH_K59A 0.69 VH_W99A 0.00 VH_D100A 0.28WT #2 1.21 VH_Y101A 0.00 VH_G102A 0.00 VH_G103A 0.00 VH_Y104A 0.00VH_F105A 0.00 VH_D106A 0.24 VH_V107A 1.08 VL_R24A 0.01 VL_A25G 0.09VL_S26S 0.36 VL_Q27A 0.03 VL_D28A 0.00 VL_I29A 0.02 VL_T30A 1.00 VL_N31A0.48 VL_Y32A 0.00 VL_L33A 0.08 WT #3 0.94 VL_N34A 0.07 VL_Y50A 0.00VL_T51A 0.80 VL_S52A 0.12 VL_R53A 0.02 VL_L54A 0.10 VL_H55A 0.33 VL_Q89A0.01 VL_Q90A 0.00 VL_G91A 0.00 VL_D92A 0.00 VL_T93A 0.64 VL_L94A 0.00VL_P95A 0.00 VL_W96A 0.00 VL_T97A 0.01

TABLE 21 Binding affinity for 20E6_H0.2aL1_hIgG1 antibody to freeLAP-TGFβ1 complex, the soluble GARP-LAP-TGFβ1 complex, andECR3E-LAP-TGFβ1 complex Sample K_(D,1) (nM) k_(on,1) (×10⁶ M⁻¹s⁻¹)k_(off,1) (×10⁻³ s⁻¹) K_(D,2) (nM) k_(on,2) (×10⁶ M⁻¹s⁻¹) k_(off,2)(×10⁻³ s⁻¹) Fc-LAP 16.16 ± 0.01 2.51 ± 0.25 40.58 ± 1.81 0.96 ± 0.090.96 ± 0.04 0.92 ± 0.07 ECR3E-LAP No binding — — — — — sGARP-LAP 18.71 ±0.05 2.77 ± 1.01 51.81 ± 6.84 — — —

Example 17: Competition Between Humanized 20E6 and Other Anti-LAPAntibodies for Binding to Soluble Human GARP-LAP-TGFβ1 Complex

Murine 28G11, 16F4, and MHG8 antibodies bind tightly to a solublecomplex consisting of the extracellular domain of human GARP and humanLAP-TGFβ1 (sGARP-LAP-TGFβ1). It was observed that 20E6_H0.2aL1_hIgG1antibody also binds tightly to this complex. Competition experiments, asshown in FIG. 25, were performed to compare the binding epitopes ofthese four antibodies.

A streptavidin-functionalized tip was equilibrated in binding buffer (10mM sodium phosphate, 150 mM sodium chloride, 1% (w/v) bovine serumalbumin, 0.05% (w/v) sodium azide, pH 7.4) and dipped in a 5 μg/mLsolution of biotinylated antibody (i.e., either 20E6_H0.2aL1_hIgG1antibody, murine 16F4 antibody, murine 28G11 antibody, or MHG8 antibody)in binding buffer for 30 seconds to load the tip with antibody. Theantibody-loaded tip was then washed in binding buffer and placed in asolution containing 24 nM of the sGARP-LAP-TGFβ1 complex. The antigenwas allowed to bind to antibody for 5 minutes (association phase), andthen the tip was moved to wells containing binding buffer alone, or 24nM of unmodified antibody. Binding of the second, unmodified antibodywas assessed by the signal change after 5 minutes of incubation.

As shown in Table 22, all four antibodies blocked binding of the sameantibody, as expected. The 20E6_H0.2aL1_hIgG1 (h12_hIgG1) antibodycompeted for binding with 28G11 antibody, but not 16F4 antibody or MHG8antibody.

TABLE 22 Competition binding data of anti-LAP antibodies to solublehuman GARP-LAP-TGFβ1 complex MHG8 16F4 h12_hIgG1 28G11 MHG8 0.00 0.300.25 0.28 16F4 0.46 0.00 0.43 0.43 h12_hIgG1 0.07 0.08 0.00 0.00 28G110.29 0.20 −0.01 0.00

Example 18: Optimized 7H4 Variant Antibodies

This Example describes the optimization of antibody 7H4 (e.g., removalof potential liability sites in the heavy chain). Specifically, position55 in the heavy chain of 7H4, which is located in the CDR2, is apotential isomerization site. To remove this potential liability site,the aspartic acid at position 55 is mutated to an amino acid other thanaspartic acid, for example, glycine, alanine, or glutamic acid, asdescribed in Table 23. The sequences are provided in Table 34.

These 7H4 variant antibodies can be tested for various functions (e.g.,binding to human LAP-TGFβ1, inhibition of TGFβ1 activation, binding toimmune cells) using the methods described herein.

TABLE 23 Multiple variants of 7H4 antibody sequences and antigen bindingfragments SEQ ID Name Description 221 7H4_HC (hyb) Murine 7H4 heavychain sequence 222 7H4_LC (hyb) Murine 7H4 light chain sequence 2317H4_HCDR2 (D55G) Murine 7H4 heavy chain CDR2 with potentialisomerization site removed (D55G) 232 7H4_HCDR2 (D55A) Murine 7H4 heavychain CDR2 with potential isomerization site removed (D55A) 2337H4_HCDR2 (D55E) Murine 7H4 heavy chain CDR2 with potentialisomerization site removed (D55E) 234 7H4_VHmut#1 (D55G) Murine 7H4heavy chain variable region with potential isomerization site removed(D55G) 235 7H4_VHmut#2 (D55A) Murine 7H4 heavy chain variable regionwith potential isomerization site removed (D55A) 236 7H4_VHmut#3 (D55E)Murine 7H4 heavy chain variable region with potential isomerization siteremoved (D55E) 224 7H4_VL Murine 7H4 light chain variable regionsequence 237 7H4_HCmut#1 (D55G) Murine 7H4 heavy chain with potentialisomerization site removed (D55G). 238 7H4_HCmut#2 (D55A) Murine 7H4heavy chain with potential isomerization site removed (D55A). 2397H4_HCmut#3 (D55E) Murine 7H4 heavy chain with potential isomerizationsite removed (D55E).

Example 19: Cryo-EM Structure of Humanized 20E6 in Complex withLAP-TGFβ1

This Example describes the identification of the epitope on LAP-TGFβ1 towhich humanized 20E6 binds, as well as the paratope of humanized 20E6,by single particle cryo electron microscopy (SP-Cryo-EM).

Sample and Grids Preparation. Humanized 20E6 mAh (20E6_H0.2aL1_hIgG1)and Fab were generated as described in Example 11. Human biotinylatedLAP-TGFβ1-Fc and GARP-LAP-TGFβ1 were generated as described in Example16. Human LAP-TGFβ1 was purchased from R&D and supplied in phosphatebuffered saline (PBS) buffer (10 mM sodium phosphate, 150 mM sodiumchloride, pH 7.4) containing 50% glycerol. Several different sampleswere prepared, with the different proteins at different concentrations,ratio, and incubation time. The following five samples were used togenerate the final reconstruction:

-   -   1) Sample A: 20 microliters (also referred to as μl or ul) of        LAP-TGFβ1-Fc (10.3 micromolar (μM or uM) in PBS (10 mM sodium        phosphate, 150 mM sodium chloride, pH 7.4)) were mixed with 4 ul        of humanized 20E6 (26 pM in PBS) for a final solution of 4.25 μM        LAP-TGFβ1-Fc and 4.3 μM humanized 20E6-Mab (1 Mab per dimer        ratio); the mixture was left on ice for 45 min and then used to        prepare grids.    -   2) Sample B: 1:5 dilution of Sample A: 4 μl Sample A+16 μl HEPES        buffered saline (HBS; 20 mM Hepes, 150 mM NaCl, pH 7.0).    -   3) Sample C: 8 μl of LAP-TGFβ1-Fc (10.3 μM in PBS) were mixed        with 8 μl of humanized 20E6 (8.6 μM in PBS) for a final solution        of 2.34 μM LAP-TGFβ1-Fc and 4.7 μM of humanized 20E6-Fab; the        mixture was left on ice for 30 min then diluted 1:1 with HBS.    -   4) Sample D: LAP-TGFβ1 in PBS with 50% glycerol was buffer        exchanged into a no-glycerol buffer (i.e., PBS), and complexed        in a 2 dimer: 1 Mab ratio with humanized 20E6-Mab, then        concentrated to 30 μM (for HDX studies). For cryo-EM studies,        the sample was diluted 10-fold with PBS.    -   5) Sample E: 1.2 μl of GARP-LAP-TGFβ1 (19.3 μM in PBS) was mixed        with 1.3 μl of humanized 20E6-Fab (8.6 μM in PBS) and 2.6 μl of        HBS buffer for a final solution of 4.4 μM GARP-LAP-TGFβ1 and 2.2        μM humanized 20E6-Fab (1 Fab per 2 dimers of GARP-LAP-TGFβ1).        The mixture was left for 30-60 mins on ice and diluted 1:10 with        HBS buffer.

Grids (C-flat carbon on gold, 300 mesh, 1.3/1.2) were prepared using aVitrobot Mark 4 (ThermoFisher) using standard procedures: grids wereglow discharged using a Pelco easyGlow unit (Ted Pella, Inc.) with thefactory suggested values for plasma cleaning (0.39 mbar, lower level 15mA, hold 10″, glow 30″). The Vitrobot was set with a chamber humiditybetween 90-100%; a chamber temperature of 4° C.; a blot time of 3 sec; await time of 0 sec; a blot force of 0. Three (3) μl of sample wereapplied to the grid, blotted, and then plunged into a liquid ethanebath; the frozen grid was then transferred to liquid nitrogen (LN2) andkept at LN2 temperature for all subsequent steps (clipping, transferringto the microscope cassette, and data collection).

Data Collection and Structure Determination. All data sets werecollected on a ThermoFisher Titan Krios G3 equipped with a Gatan K3Direct Electron Detector. Data collection was done using the GatanLatitude software. Five data sets (one per each prepared sample) werecollected. Table 24 summarizes the microscope and camera parameters usedfor data collection, and the total number of movies collected for eachsample. The entire data collection (for the 5 samples) spanned twoweeks.

TABLE 24 Cryo-EM details for 20E6 humanized antibody and Fab Detector #of pixel size Dose Defocus Sample Movies Voltage Magnification (Å)(e⁻/Å²) (μm) A) LAP-TGFβ1-FC + 2690 300 81,000 1.07 62.5 −0.8 to −1.8Humanized 20E6-Mab, 1 Mab/dimer B) LAP-TGFβ1-FC + 1994 300 81,000 1.0762.5 −1.2 to −1.6 Humanized 20E6-Mab, 1 Mab/dimer (1:5 dilution) C)LAP-TGFβ1-FC + 2647 300 81,000 1.07 62.5 −1.2 to −1.6 Humanized20E6-Fab, 2 Fab/Dimer D) LAP-TGFβ1 + 4545 300 81,000 1.07 62.5 −0.8 to−1.8 Humanized 20E6-Mab (1:10 dilution) E) GARP-LAP- 9366 300 81,0001.07 62.5 −0.8 to −1.8 TGFβ1 + Humanized 20E6-Fab (1 Fab/1 dimer) 1:10dilutionData processing and map reconstruction. The entire data processing andmap reconstruction were carried out with Cryosparc V2 (StructuraBiotechnology Inc., Toronto, Canada; Punjani et al. Nature Methods 2017;14:290-6; Brubaker et al. IEEE Trans Pattern Anal Mach Intell 2017;39:706-18). Initial stages of the processing pipeline (Movie alignment,CTF estimation, particle picking, and 2D classes determination) wascarried out individually for each data set. Cleaned particles from theindividual data sets were merged together in three stages.

-   -   1) Particles from the first three data sets (samples A-C), were        merged and processed together but the best map was only at 5 Å.    -   2) Using particles from sample D it was possible to generate a        3.8 Ang map. Templates from sample D were then used for a new        particle picking for samples A-C. Selected particles were then        subjected to one round of 2D classification and merged with the        best particle set from Sample D for a total of 533,297        particles. After one round of 2D classification, the best        classes (436,918 particles) were used to run a homogeneous        refinement job. The resulting map was subjected to one round of        non-uniform refinement (NU-refinement) which resulted in a map        with a resolution of 3.5 A.    -   3) A set of ˜1,2M particles from sample E was merged with the        latest (best) set from 2. After 2 rounds of 2D classification,        the best 2D classes (864,958 particles) were used to calculate a        map that (after NU refinement) had a nominal resolution of 3.4        Å.

Another 2 rounds of 2D classification on the 864,958 particles were thenused to generate a set containing 802,256 particles and a non-uniform(NU_-refined map at 3.3 Å. Throughout the whole process, visualinspection of the resulting maps (density improvement, continuity indensity, lack of (or reduction on) preferred orientation artifacts) wasused to decide subsequent steps. The latest map clearly pointed to thefact that, while two Fabs/LAP-TGFβ1 dimer are present, one is much morebetter defined than the other (FIG. 26A), and thus particle subtractionand local refinement procedures were applied. The resulting final maphad a nominal resolution of 3.1 Å. This map was used to build and refinethe final model. FIGS. 26B and 26C show the FSC plot and the Guinierplot for the final map.

Model building and refinement. All model building and refinement werecarried out using COOT (Emsley et al. Acta Crystallogr D-BiologicalCrystallography 2010; 66:486-501) and PHENIX (Afonine et al. ActaCrystallogr D Struct Biol 2018; 74 6:531-44). LAP-TGFβ1 coordinates wereobtained from PDB entry 3RJR, and Fab coordinates were obtained from ahomology model generated using MOE 2019.0101 (Chemical Computing GroupULC). LAP/TGFβ1 and the Fab model were initially positioned into the mapas rigid bodies using COOT, and the density was used to rebuild some ofthe loops and assign the correct sequences. The PHENIX real spacerefinement module was carried out to optimize the model geometry. Table25 summarizes model refinement and statistics:

TABLE 25 Model refinement and statistics for the cryo-EM of humanized20E6 Fab and LAP-TGFβ1 Symmetry Imposed C1 Particle used 802,256 Mapresolution (Å) 3.1 FSC threshold 0.143 Map Resolution Range (Å) 3-19Refinement Map sharpening B-factor (Å²) 87.5 Model composition NonHydrogen Atoms  8,757 Protein residues  1,110 CC_mask: 0.655 CC_volume:0.638 CC_peaks: 0.525 rmsd (bonds) (Å): 0.01 rmsd (angles) (°): 0.98All-atom clashscore 5.41 Ramachandran plot: outliers: 0.00% allowed:7.16% favored: 92.84% Rotamer outliers: 0.20%

The final model contained two chains for the LAP-TGFβ1 dimer (chains Aand B, each one containing residues 1-61+70-208+216-241+250-361;numbering for antigen assumes absence of signal peptide, i.e. Leu 1=Leu30 in complete sequence); two chains (heavy chain (VH), residues 1-221and light chain (VL), residues 2-214) for the humanized 20E6-Fab. Onesugar moiety (NAG) was modeled at one of the glycosylation sites (AsnA53); for all the other possible glycosylation positions (A107, A147,B53, B107, and B147) the density was not sufficient to warrant the sugaraddition).

Structural Analysis. The structure of the LAP-TGFβ1 dimer in complexwith humanized 20E6-Fab was determined to 3.1 Å resolution. The qualityof the cryo-EM map was such that assignment of side chains for both theantigen and the Fab was unequivocal. At the antigen-antibody interface,the quality of the map is comparable to that of x-ray derived electrondensity maps calculated at comparable resolution (FIG. 27A: cryo-EM mapat 3.1 Å resolution; FIG. 27B: electron density for Protein Data Bank(PDB) entry 5jxe at 2.9 Å resolution).

Due to the intrinsic characteristics of cryo-EM maps (Cardone G, et. Al,J Struct Biol. 2013; 184:226-236), there is a clear gradient between theLAP-TGFβ1:Fab interface and the rest of the molecule. A lower leveldensity is present for a second, symmetrically bound Fab, which could bedocked into the final map (FIG. 26A).

The humanized 20E6-Fab paratope and LAP-TGFβ1 epitope residues thatcomprise the interaction interface are shown in FIGS. 28A and 28B, andsummarized in Table 26. The interface is made up of van der Waals andelectrostatic interactions, and corresponds to ˜800 Å² of buriedsurface, as calculated by Protein interfaces, surfaces and assemblies(PISA), (Krissinel et al, J Mol Biol 2007; 372:774-97). The epitope isformed by residues A31-P40 from chain A (LAP residues) and Y340-R343 andR274-K280 from chain B (TGFβ1 residues). The fact that both LAP andTGFβ1 residues are required for interactions with humanized 20E6-Fabexplains why the antibody is specific for the closed form of theLAP-TGFβ1 complex, and does not bind to empty LAP or to mature TGFβ1.The paratope is formed by light chain (VL) residues: T30, Y32, Y49-Y50,R53, G91-L94, W96, and heavy chain (VH) residues W33, R50, I58-K59, W99,and Y101-G103. Epitopes determined by the cryo-EM analysis are inagreement with the hydrogen deuterium exchange mass spectrometry(HDX-MS) analysis described in Example 22. Paratopes are in agreementwith residues identified by alanine scanning experiments described inExample 14. See also FIGS. 28C-D.

TABLE 26 Epitope and paratope of LAP-TGFβ1 and humanized 20E6-Fab* VHTGFβ1 VL R274 T30 G278 T30 W279 Y50, Y50 K280 D92 Y101 V341 Y101, W33G342 VH LAP VL A31 Y49, R53 Y104, Y104 S32 Y49, Y50 G102 P33 Y32 P34 Y32G102, W99, S35 G91, Y32 Q36 D92 G37 L94, D92 R50, K59, W33 E38 L94, W96*Residues from humanized 20E6-Fab VH or VL that interact with LAP-TGFβ1residues are indicated. Hydrogen bonding interactions are indicated inbold (interaction cut-off set to 4.5 Å; hydrogen bond interactionscut-off set to 3.5 Å).

Example 20: Cryo-EM Structure of Humanized 28G11 in Complex withLAP-TGFβ1

The structure of humanized 28G11 Fab in complex with human LAP-TGFβ1 wasdetermined by cryo-EM to identify the epitope on LAP-TGFβ1 to which theantibody binds, and the paratope of humanized 28G11-Fab.

Sample and Grids Preparation. Humanized 28G11 mAb (28G11_H2bL3a_hIgG1,which has heavy and light chain variable region sequences of SEQ ID NOs:43 and 53, respectively) and GARP-LAP-TGFβ1 were generated as describedin Examples 9 and 16, and supplied in PBS buffer (10 mM sodiumphosphate, 150 mM sodium chloride, pH 7.4). The sample used for cryo-EMexperiments was prepared by mixing 2.0 μl of GARP-LAP-TGFβ1 (38.7 μM),0.5 μl of humanized 28G11 (77.3 μM), and 17.5 μl of HBS buffer (20 mMHepes, 150 mM NaCl, pH 7.0) for a final concentration of 3.87 μM forGARP-LAP-TGFβ1 and 1.95 μM for humanized 28G11. The sample was furtherdiluted 1:1 with HBS before preparing the grids. The complex containingGARP (rather than LAP-TGFβ1 alone) was used to disrupt the preferredorientation issues observed in data collected from samples of humanized28G11:LAP-TGFβ1.

Grids (C-flat carbon on gold, 300 mesh, 1.3/1.2) were prepared using aVitrobot Mark 4 (ThermoFisher) using standard procedures. Grids wereglow discharged using a Pelco easyGlow unit (Ted Pella, Inc.) with thefactory suggested values for plasma cleaning (0.39 mbar, lower level 15mA, hold 10″, glow 30″). The Vitrobot was set with a chamber humiditybetween 90-100%; a chamber temperature of 4C°; a blot time of 3 sec; await time of 0 sec; a blot force of 0. Three (3) μl of sample wereapplied to the grid, blotted, and then plunged into a liquid ethanebath; the frozen grid was then transferred to liquid nitrogen (LN2) andkept at LN2 temperature for all subsequent steps (clipping, transferringto the microscope cassette, and data collection).

Data Collection and Structure Determination. The data set was collectedon a ThermoFisher 300 KeV Titan Krios G3 equipped with a ThermoFisherFalcon 3 Direct Electron Detector. Data collection was done using theThermoFisher EPU software. 4267 movies were collected at a nominalmagnification of 75,000×; the defocus range was set to be between −1.4and −2.0 μm. The detector pixel size was 1.06 Å and the dose was 37.74e⁻/Å².

Data processing and map reconstruction. The entire data processing andmap reconstruction was carried out with Cryosparc V2. The initialparticle picking identified 2.9M particles. After two 2D classificationjobs, about 620K particles were used to calculate an initial map(nominal resolution 3.81 Ang). The particle stack was further cleaned upusing two more 2D classifications, and the resulting set of particles(505,582 particles) were used to generate a map that after aNU-refinement had a nominal resolution of 3.48 Ang. Local (masked)refinement was then used to improve the resolution at theepitope-paratope interface. The result of the local refinement (afterparticle subtraction) was a 3.38 Ang map in which the details at theinterface were greatly improved. This map was used to build the model.

Model building and refinement. All model building and refinement werecarried out using COOT. The complex between LAP-TGFβ1 and humanized 20E6Fab was used as the starting model; LAP-TGFβ1 and humanized 28G11-Fabwere initially positioned into the map as rigid bodies using COOT, andthe density was used to rebuild some of the loops and assign the correctsequences. The PHENIX real space refinement module was carried out tooptimize the model geometry. Table 27 summarizes the model refinementand statistics:

TABLE 27 Model refinement and statistics of humanized 28G11-Fab andLAP-TGFβ1 Symmetry Imposed C1 Particle used 505,582 Map resolution (Å)3.4 FSC threshold 0.143 Map Resolution Range (Å) 3.0-14.0 Refinement Mapsharpening B-factor (Å²) 105.6 Model composition Non Hydrogen Atoms 8,960 Protein residues  1,113 CC_mask: 0.716 CC_volume: 0.702 CC_peaks:0.628 rmsd (bonds) (Å): 0.007 rmsd (angles) (°): 0.81 All-atomclashscore 19.8 Ramachandran plot: outliers: 0.00% allowed: 15.58%favored: 84.42% Rotamer outliers: 0.20%

The final model contained two chains for the LAP-TGFβ1 dimer (chains Aand B, each one containing residues 1-61+70-208+216-241+250-361;numbering for antigen assumes absence of signal peptide, i.e. Leu 1=Leu30 in complete sequence) and one molecule (heavy chain (VH), residues1-221 and light chain (VL), residues 2-214) for the humanized 28G11-Fab.One sugar moiety N-acetylglucosamine (NAG) was modeled at one of theglycosylation sites (Asn A53); for all the other possible glycosylationpositions (Asn A107, Asn A147, Asn B53, Asn B107, and Asn B147) thedensity was not sufficient to warrant the sugar addition).

Structural Analysis. The structure of the LAP-TGFβ1 dimer in complexwith humanized 28G11-Fab was determined to 3.4 A resolution. The qualityof the cryo-EM map was such that assignment of side chains for both theantigen and the Fab was unequivocal.

The humanized 28G11-Fab paratope and LAP-TGFβ1 epitope residues thatcomprise the interaction interface are shown in FIGS. 29A and 29B, andsummarized in Table 28. See also FIGS. 29C-E. The interface is made upof van der Waals and electrostatic interactions, and corresponds to ˜800Å² of buried surface, as calculated by PISA. The epitope is formed byresidues A31-E38 from chain A (LAP residues) and G342-K344 and G278-W281from chain B (TGFβ1 residues). The fact that both LAP and TGFβ1 residuesare required for interactions with h20E6-Fab explains why the antibodyis specific for the closed form of the LAP-TGFβ1 complex, and does notbind to empty LAP or to the mature TGFβ1. The paratope is formed bylight chain (VL) residues: Y32, Y49-Y50, R53, and G91-L94, and heavychain (VH) residues W33, F50-N53, Q56, and Y101-Y106.

TABLE 28 Epitope and paratope of LAP-TGFβ1 and 28G11-Fab* VH TGFβl VLG278 S30 W279 Y50 K280 D92 W281 Y32 Q56, W33 G342 R52, Q56 R343 Q56 K344VH LAP VL A31 Y49, R53 Y106, Y106 S32 Y49, Y50, R53 G104 P33 Y32, Y50P34 Y32, Y32 Y101, G104 S35 Y32, G91 Q36 D92 G37 D92, T93, L94 F50, R52,R52, Y33, E38 L94 Y33 *Residues from 28G11-Fab VH or VL that interactwith LAP-TGFβ1 residues are indicated. Hydrogen bonding interactions areindicated in bold (interaction cut-off set to 4.5 Å; hydrogen bondinteractions cut-off set to 3.5 Å).

Example 21: Cryo-EM Structure of Murine 22F9 in Complex with LAP-TGFβ1

The structure of murine 22F9-Fab (referred to in the Example as 22F9) incomplex with human LAP-TGFβ1 was determined by SP-Cryo-EM to identifythe epitope on LAP-TGFβ1 to which the antibody binds, and the paratopeof 22F9-Fab.

Sample and Grids Preparation. The murine 22F9 mAh used in thisexperiment was 22F9_N54Q_D102A_mIgG2a, which has heavy and light chainvariable region sequences of SEQ ID NOs: 248 and 249, respectively.Human LAP-TGFβ1 was purchased from R&D and supplied in PBS buffer (10 mMsodium phosphate, 150 mM sodium chloride, pH 7.4) containing 50%glycerol. The sample was prepared as follows: LAP-TGFβ1 in PBS with 50%glycerol was buffer exchanged into a no-glycerol buffer (i.e., PBS), andcomplexed in a 1 dimer: 1 Mab ratio with 22F9-Mab, then diluted 10-foldwith PBS.

Grids (C-flat carbon on gold, 300 mesh, 1.3/1.2) were prepared using aVitrobot Mark 4 (ThermoFisher) using standard procedures. Specifically,grids were glow discharged using a Pelco easyGlow unit (Ted Pella, Inc.)with the factory suggested values for plasma cleaning (0.39 mbar, lowerlevel 15 mA, hold 10″, glow 30″). The Vitrobot was set with a chamberhumidity between 90-100%; a chamber temperature of 4C°; a blot time of 3sec; a wait time of 0 sec; a blot force of 0. Three (3) pi of samplewere applied to the grid, blotted, and then plunged into a liquid ethanebath. The frozen grid was then transferred to liquid nitrogen (LN2) andkept at LN2 temperature for all subsequent steps (clipping, transferringto the microscope cassette, and data collection).

Data Collection and Structure Determination. The data set was collectedon a ThermoFisher 300 KeV Titan Krios G3 equipped with a Gatan K3 DirectElectron Detector. Data collection was done using Gatan Latitudesoftware. 3741 movies were collected at a nominal magnification of81,000×; the defocus range was set to be between −0.8 and −1.8 μm. Thedetector pixel size was 1.07 Å and the dose was 62.5 e⁻/Å².Data processing and map reconstruction. The entire data processing andmap reconstruction was carried out with Cryosparc V2. Initial processingidentified 2.9M particles. Several 2D classification jobs were run toclean up the particle stack and remove outliers. At the end, 522,208particles were used in a homogeneous refinement job which yielded a 3.68Ang map. Non-uniform refinement yielded a 3.43 Ang map that was used tobuild the model. The 2D classes clearly showed the presence of a2Mab:2TGFβ1 complex, confirming the stoichiometry suggested by SEC-MALexperiments (FIGS. 30A and 30B).Model building and refinement. All model building and refinement werecarried out using COOT and PHENIX (Afonine et al. Acta Crystallogr DStruct Biol 2018; 74 6:531-44). The complex between LAP/TGFβ1 andhumanized 20E6 was used as starting model; LAP-TGFβ1 and humanized20E6-Fab were initially positioned into the map as rigid bodies usingCOOT, and the density was used to rebuild some of the loops and assignthe correct sequences. The PHENIX real space refinement module wascarried out to optimize the model geometry. Table 29 summarizes themodel refinement and statistics:

TABLE 29 Model refinement and statistics of humanized 22F9-Fab andLAP-TGFβ1 Symmetry Imposed C2 Particle used 522,208 Map resolution (Å)3.4 FSC threshold 0.143 Map Resolution Range (Å) 3-14 Refinement Mapsharpening B-factor (Å²) 116.0 Model composition Non Hydrogen Atoms11926 Protein residues 1530 CC_mask: 0.689 CC_volume: 0.680 CC_peaks:0.615 rmsd (bonds) (Å): 0.007 rmsd (angles) (°): 0.91 All-atomclashscore 25.1 Ramachandran plot: outliers: 0.40% allowed: 18.1%favored: 81.5% Rotamer outliers: 1.95%

The final model contained two chains for the LAP-TGFβ1 dimer (chains Aand B, each one containing residues 1-61+70-208+216-241+250-361;numbering for antigen assumes absence of signal peptide, i.e., Leu 1=Leu30 in complete sequence); two chains (heavy chain (VH), residues 1-221and light chain (VL), residues 2-214) for the 22F9-Fab. One sugar moiety(NAG) was modeled at glycosylation sites Asn A53 and Asn B53); thedensity was not sufficient to warrant the sugar addition for all otherpossible glycosylation positions (A107, A147, B107, and B147).

Structural Analysis. The structure of the LAP-TGFβ1 dimer in complexwith the 22F9-Fab was determined to 3.4 A resolution. The quality of thecryo-EM map was such that assignment of side chains for both the antigenand the Fab was unequivocal.

The 22F9-Fab paratope and LAP-TGFβ1 epitope residues that comprise theinteraction interface are shown in FIGS. 31A and 31B and summarized inTable 30.

The interface is made up of van der Waals and electrostaticinteractions, and corresponds to ˜900 Å² of buried surface, ascalculated by PISA. The epitope is formed by residues S35-P43 from chainA (LAP residues) and D272-K275, K280-H283, and Y340 from chain B (TGFβ1residues).

The 22F9 paratope is formed by light chain (VL) residues: Y36, Y53,L58-S60, and R98-Y100, and heavy chain (VH) residues S31-W33, H52, andY98-D106. See FIGS. 31C-E.

TABLE 30 Epitope and paratope of LAP-TGFβ1 and 22F9-Fab* VH TGFβ1 VLD272 Y53 D100, Y104, D106 R274 Y53, E59 K275 Y53, L58, E59, S60 Y32,Y98, Y99, D100, K280 Y101, D106 Y101 W281 Y101 H283 Y101 Y340 VH LAP VLS31, Y32 S35 W33, W33, Y32, H52 Q36 Y99, Y99, W33, W33, G37 E38 M50,Y101, Y99 V39 Y99 P41 Y100, R96, E97, L98 G42 R96, R96, Y36, Y36 P43T31, Y36, R96 *Residues from 22F9-Fab VH or VL that interact withLAP-TGFβ1 residues are indicated. Hydrogen bonding interactions areindicated with bold type (interaction cut-off set to 4.5 Å; hydrogenbond interactions cut-off set to 3.5 Å).

Example 22: Analysis of a Humanized 20E6 Antibody by Hydrogen DeuteriumExchange Mass Spectrometry

Contact areas between the humanized anti-LAP antibody 20E6_H0.2_hIgG4mut(referred to as “humanized 20E6” in this Example) and human LAP-TGFβ1were determined by hydrogen deuterium exchange mass spectrometry(HDX-MS). HDX-MS measures the exchange of deuterium with hydrogen intothe amide backbone of the protein. One factor influencing the exchangerate is the hydrogen's exposure to solvent. Comparison of the exchangelevels in the antigen when the antibody is bound can identify regions ofthe protein where the antibody is binding.

Materials

-   -   Human LAP-TGFβ1 protein was purchased from R&D Systems and        consists of an N-terminal 249 aa latency-associated peptide        (LAP) and a C-terminal 112 aa mature TGFβ1 protein. The protein        was buffer exchanged and concentrated to 40 μM in 10 mM sodium        phosphate, 150 mM sodium chloride, pH 7.4.    -   Humanized anti-LAP-TGFβ1 antibody (20E6_H0.2_hIgG4mut) was        generated as described in Example 11. The antibody was diluted        from 7.1 mg/mL to 5.8 mg/mL, equivalent to 40 μM.

Liquid Chromatography-Mass Spectrometry

A Waters Synapt G2Si Quadrupole Time-of-flight (TOF) mass spectrometerwas used. For peptide identification and measurement of deuteriumlabeled samples, the mass spectrometer was set to acquire one full scanMS data (low energy) and one MS(e) data (high energy) in the TOF-onlymode. The scan time was set to 0.4 seconds. Ramp trap collision energywas from 15 to 45 volts.

The liquid chromatography system was a Waters nanoAcquity binary pumpfor the analytical column gradient and auxiliary pump for sampledigestion and loading. For sample digestion and loading, the buffer usedwas 100% water and 0.1% formic acid at a flow rate of 100 μL/minute. Forthe analytical gradient, the buffers were Buffer A (0.1% formic acid inwater) and Buffer B (0.1% formic acid in acetonitrile).

The gradient was at 40 μL/minute from 5% B to 35% B in 9 minutes,followed by a ramp to 85% B in one minute, a wash of 85% B for oneminute, and a re-equilibration at 5% B for one minute. The column wasthen washed by cycling the gradient between 5% and 95% B, four timeswith one minute at each step, followed by a final equilibration at 5% Bfor one minute. The trapping column was a Waters Vanguard BEH C18 1.7 μmGuard Column and the analytical column was a Waters BEH C18, 1.7 μm 1×50mm column.

Sample handling for the deuterium labeling was done by a Waters HDX unitwhich consists of Leaptec H/D-X PAL system and a Waters HDX chamber forcolumn cooling. The labeling sample tray was set to a temperature of 10°C., the quenching tray was set to 1.5° C., and the trap and analyticalcolumn chamber was set to 1.5° C. The immobilized protease typeXIII/pepsin column (w/w, 1:1) from NovaBioassays was kept at 20° C. inthe enzyme column chamber.

Deuterium Labeling

Human LAP-TGFβ1 was mixed with humanized 20E6 to final concentrations of20 μM for human LAP-TGFβ1 and 10 μM for humanized 20E6. An unboundcontrol was prepared by incubating human LAP-TGFβ1 in 10 mM sodiumphosphate, 150 mM sodium chloride, pH 7.4. The antibody bound sample andthe unbound control were incubated at room temperature for one hourbefore beginning the labeling experiment.

To deuterium label the samples, 6 μL of sample was mixed with 54 μL of10 mM sodium phosphate, 150 mM sodium chloride in deuterium oxide pD7.4. Labeling time points were 0, 10, 60, 600, 6000, and 14,400 seconds.After each time point, 50 μL of the labeling mixture was added to 50 μLof cold quench buffer (500 mM tris(2-carboxyethyl)phosphine (TCEP) inphosphate buffer, pH 2.5). After mixing once, 90 μL was then injectedinto the column cooling chamber where the sample was passed over theprotease type XIII/pepsin column and the resulting peptides loaded ontothe trapping column. After 4 minutes, a valve switch took the proteasetype XIII/pepsin column out of line. The trap was then switched in-linewith the analytical column and the analytical gradient and the massspectrometer data acquisition was started. Each time point was acquiredin duplicate.

Data Analysis

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data wasacquired of unlabeled bound and unbound samples in quadruplicate, anddatabase searches with ProteinLynx Global Server 3.0 (WatersCorporation) were performed to verify successful digestion of theproteins and to generate a list of peptides from the dual-enzymedigestion. The protein database used was human LAP-TGFβ1 combined with arandomized human LAP-TGFβ1 sequence to reduce false identification.

Mass spectrometry (MS) data from the deuterium labeling experiment wasprocessed by DynamX (version 3.0.0, Waters Corporation). For eachpeptide, the mass, retention time, and charge state selected by thesoftware were verified manually.

Results

The human LAP-TGFβ1 peptides protected by humanized 20E6 are shown in anH/D Difference Plot (FIG. 32).

Example 23: Epitope Mapping of Murine 28G11, 22F9, 20E6, and 2F8Antibodies by HDX-MS

Contact areas between murine anti-LAP-TGFβ1 antibodies 28G11, 22F9,20E6, and 2F8 and human LAP-TGFβ1 were determined by HDX-MS, asdescribed below.

Methods

Materials used were as follows:

-   -   Human LAP-TGFβ1 protein was purchased from R&D Systems and        consists of an N-terminal 249 aa latency-associated peptide        (LAP) and a C-terminal 112 aa mature TGFβ1 protein. The protein        was buffer exchanged and concentrated to 40 μM in 10 mM sodium        phosphate and 150 mM sodium chloride, pH 7.4.    -   Murine 28G11_mIgG2a, murine 22F9_mIgG2a, and murine 20E6_mIgG2a        were diluted to 40 μM. The heavy and light chain variable region        sequences of 28G11(hyb), 22F9 (hyb), and 20E6 (hyb) (provided in        Table 34) were fused to an mIgG2a constant Murine 2F8 (IgG1) was        purchased from BioLegend and concentrated from 0.5 mg/mL to 40        μM.

Liquid chromatography-mass spectrometry was performed in the mannerdescribed in Example 22, except that, in the sample handling fordeuterium labeling, the labeling sample tray was set to a temperature of25° C. instead of 10° C. Deuterium labeling was also performed asdescribed in Example 22, except that human LAP-TGFb1 was mixed with theantibody to final concentrations of 20 μM for human LAP-TGFβ1 and 20 μMfor the antibody. Data analysis was performed as described in Example22.

Results

Human LAP-TGFβ1 peptides protected by the antibodies are illustrated inthe H/D Difference Plots shown in FIG. 33. Binding epitopes for 28G11,22F9, and 20E6 covered four regions in the LAP-TGFβ1 protein: amino acidresidues 14-25 (RKRIEAIRGQIL, region 1; SEQ ID NO: 250), 30-39(LASPPSQGEV, region 2; SEQ ID NO: 251), 278-286 (GWKWIHEPK, region 3;SEQ ID NO: 252), and 340-346 (YVGRKPK, region 4; SEQ ID NO:253). Regions1 and 2 are in the LAP domain, and region 3 and 4 are in the matureTGFβ1 domain.

Differences in the degree of deuterium exchange protection were detectedamong the antibodies. For example, in comparing 20E6 with 28G11 and22F9, 20E6 showed no deuterium exchange protection at region 1, while28G11 and 22F9 had detectable changes upon antibody binding(approximately 1.3 and 1.9 Da, respectively). Slight differences indeuterium exchange were also observed for regions 2 and 3. Specifically,in region 2, a 6 Da difference was detected for 20E6, but the differencefor 28G11 was only 4 Da. In region 3, a 4 Da difference was detected for20E6, but only a 2 Da difference was detected for 28G11. Notably, theinteraction at region 1 detected by HDX was not observed in the cryo-EMstructure, suggesting that deuterium exchange protection at region 1 isnot due to direct antibody:antigen binding, but from changes in localsolution dynamics as a result of slightly different binding interactionsbetween these antibodies and LAP-TGFβ1. Taken together, the HDX data areconsistent with the cryo-EM structures.

It was observed that antibody 2F8 bound to residues 205-225 (amino acidsVDINGFTTGRRGDLATIHGMN; SEQ ID NO: 254).

The amino acid sequences of murine and human LAP-TGFβ1 are 89%identical. Epitopes identified are all in the homologous region (FIG.34).

Example 24: Binding Stoichiometry of Human LAP-TGFβ1 or GARP-LAP-TGFβ1Complexed with a Humanized 20E6 Antibody by Size-ExclusionChromatography and Multi-Angle Light Scattering

The binding stoichiometry of human LAP-TGFβ1 or GARP-LAP-TGFβ1 complexedwith 20E6_H0.2_hIgG4mut was determined by size-exclusion chromatographyand multi-angle light scattering (SEC-MALS). Due to differences in size,human LAP-TGFβ1 alone, 20E6_H0.2_hIgG4mut alone, and the complex eluteat different times on SEC chromatogram. The MALS detector helpsdetermine the molecular weight for each detected peak. Based on themolecular weight of individual proteins and the protein complex, bindingstoichiometry can be determined.

Materials

-   -   Human LAP/TGFβ1 protein was purchased from R&D Systems and        consists of an N-terminal 249 aa latency-associated peptide        (LAP) and a C-terminal 112 aa mature TGFβ1 protein. The protein        was buffer exchanged and concentrated to 40 μM in 10 mM sodium        phosphate, 150 mM sodium chloride, pH 7.4.    -   Human GARP-LAP-TGFβ1 was generated as described in Example 16.    -   Anti-human LAP-TGFβ1 antibody (20E6_H0.2_hIgG4mut) was generated        as described in Example 11. The antibody was diluted from 7.1        mg/mL to 5.8 mg/mL equivalent to 40 μM.

Size-Exclusion Chromatography—Multi-Angle Light Scattering

Size-exclusion chromatography was performed using an Agilent 1200 HPLCconnected to a photodiode array detector and a Wyatt light scatteringdetector. Superdex 200 Increase 5/150 GL column was run at a 0.2mL/minutes flow rate using 10 mM sodium phosphate, 150 mM sodiumchloride, pH 7.4 buffer under an isocratic gradient.

Human LAP-TGFβ1 was mixed with antibody 20E6_H0.2_hIgG4mut to finalconcentrations of 5 μM for human LAP-TGFβ1 and antibody 2.5 μM for20E6_H0.2_hIgG4mut. Thirty μL of 5 μM human LAP/TGFβ1 alone, 5 μM20E6_H0.2_hIgG4mut alone, and LAP-TGFβ1:20E6 complex were analyzed usingthe SEC-MALS system including a gel filtration standard and bovine serumalbumin standard. The following samples were also analyzed: 30 μL of 7.5μM human LAP-TGFβ1 alone, 7.5 μM antibody 20E6_H0.2_hIgG4mut alone, andGARP-LAP-TGFβ1:20E6_H0.2_hIgG4mut complex.

Data Analysis

All chromatograms were plotted using ChemStation (Version A.01.08.108,Agilent Technologies) at 280 nM UV absorbance. The light scattering datawas analyzed using ASTRA (Version 6.1.2.84, Wyatt Technologies). Allpeaks were integrated cross full width at half height.

Results

SEC-MALS analysis for LAP-TGFβ1:20E6_H0.2_hIgG4mut complex andGARP-LAP-TGFβ1 complex are shown in FIGS. 35A and 35B, respectively.Based on the molecular weight of each protein and the protein complex,the binding stoichiometry for the LAP-TGFβ1 complex is 2:2 molar ratio,i.e., two copies of LAP-TGFβ1 dimer bind to two copies of20E6_H0.2_hIgG4mut antibody; the binding stoichiometry for theGARP-LAP-TGFβ1 complex is 2:1 molar ratio. In the presence of GARP, onlyone binding site in LAP-TGFβ1 dimer is able to interact with theantibody.

Example 25: Efficacy of Anti-LAP Antibodies in CT26 Syngeneic Model

This Example describes the efficacy of anti-LAP antibodies incombination with anti-PD-1 antibodies in the CT26 colorectal cancertumor model, a syngeneic model of cancer. In this experiment, variantsof the anti-LAP antibodies were used in which the Fc portion of theantibody was the IgG2a isotype rather than the isotype found in theparental hybridoma.

Briefly, 6-8 week-old Balb/c mice were subcutaneously implanted with3×10⁵ CT26 colorectal cancer cells. Tumors were grown until an averagesize of 48 mm² at which point tumor-bearing animals were randomized togroups of 10 animals each.

One set of animals was dosed intraperitoneally with either rat anti-PD-1clone RMP1-14-IgG2a at 3 mg/kg or a combination of anti-PD-1 andantibody 28G11-IgG2a at 10 mg/kg on days 0, 3, 6, 9, and 12. Animalgroups were also dosed with isotype control antibodies (rat-IgG2a and/ormouse IgG2a, not shown).

Another set of animals was dosed intraperitoneally with either ratanti-PD-1 clone RMP1-14-IgG2a at 3 mg/kg, antibody 16B4-IgG2a at 10mg/kg, or a combination of anti-PD-1 and antibody 16B4-IgG2a at 10 mg/kgon days 0, 3, 6, 9, and 12. Animal groups were also dosed with isotypecontrol antibodies (rat-IgG2a and/or mouse IgG2a, not shown).

Survival was assessed daily and tumor volumes were measured 3 times perweek by caliper using the formula V=W2×L/2. Animals were followed for 53days post dosing.

As shown in FIGS. 36A and 36B, treatment of this syngeneic model withantibody 28G11 resulted in a 5-fold increase in complete response rateover anti-PD-1 alone. In contrast, as shown in FIGS. 36C-36F, treatmentof animals with antibody 16B4 had no effect on tumor growth. In fact,treatment of animals with a combination of 16B4 and anti-PD-1 resultedin a reduction of the response rate seen with anti-PD-1 antibody alone.These data establish that the two anti-LAP antibodies 28G11 and 16B4have different functional properties in a mouse tumor model.

Example 26: Efficacy of Anti-LAP Antibodies in EMT6 Syngeneic Model

This Example describes the efficacy of anti-LAP antibodies incombination with anti-PD-1 antibodies in another syngeneic model ofcancer, i.e., the EMT6 breast cancer tumor model.

Briefly, 6-8 week-old Balb/c mice were subcutaneously implanted into theright hind flank with 3×10⁵ EMT6 breast cancer cells. Tumors were grownuntil an average size of 75 mm², at which point tumor-bearing animalswere randomized to 10 groups of 10 animals each, and dosedintraperitoneally on days 0, 3, 6, 9, 12, 15, 18, and 21 according tothe following:

TABLE 31 Antibody and dosing information for the EMT6 syngeneic modelGroup Description Dose 1 Rat IgG2a  5 mg/kg Mouse IgG2a 15 mg/kg 2 Ratanti-PD-1 RMP1-14-IgG2a  5 mg/kg Mouse IgG2a 15 mg/kg 3 28G11_IgG2a 10mg/kg Rat IgG2a  5 mg/kg 4 Rat anti-PD-1 RMP1-14-IgG2a  5 mg/kg28G11_IgG2a 10 mg/kg 5 22F9_IgG2a 10 mg/kg Rat IgG2a  5 mg/kg 622F9_IgG2a 15 mg/kg 7 Rat anti-PD-1 RMP1-14-IgG2a  5 mg/kg 22F9_IgG2a 10mg/kg 8 Rat anti-PD-1 RMP1-14-IgG2a  5 mg/kg 22F9_IgG2a 15 mg/kg 920E6_IgG2a 10 mg/kg Rat IgG2a  5 mg/kg 10  Rat anti-PD-1 RMP1-14-IgG2a 5 mg/kg 20E6_IgG2a 10 mg/kg

Survival was assessed daily and tumor volumes were measured 3 times perweek by caliper using the formula V=W2×L/2. Animals were followed for 28days post dosing. Data is graphed as mean tumor volume+/−SEM.

As shown in FIG. 37, treatment of animals with antibody 28G11 eitheralone or in combination with anti-PD-1 resulted in a statisticallysignificant reduction in tumor growth relative to isotype controlantibody or anti-PD-1 alone. Similarly, treatment of animals withantibody 22F9 (FIG. 38) and 20E6 (FIG. 39), either alone or incombination with anti-PD-1, resulted in a statistically significantreduction in tumor growth relative to isotype control antibody oranti-PD-1 alone. These data demonstrate that 28G11, 22F9 and 20E6 areall active in combination with anti-PD-1 antibody in the EMT6 mousemodel.

Example 27: Efficacy of Anti-LAP Antibodies in 4T1 Breast Cancer TumorMetastasis Model

This Example describes the efficacy of anti-LAP antibodies asmonotherapy in a model of tumor metastasis, i.e., the 4T1 breast cancertumor metastasis model.

Briefly, 1×10⁵ 4T1 breast cancer cells were implanted into the mammaryfat pad of 6-8 week-old Balb/c mice. One day after implantation, animalswere randomized to groups of 7 animals each. Animals were dosed withmouse IgG1 isotype control antibody, mouse-IgG2a control antibody,anti-TGFβ clone 1D11-IgG1, and anti-LAP antibodies 28G11 and 16B4. Allanimals were dosed intraperitoneally at 10 mg/kg on days 0, 3, 6, 9, and12. On day 29 post dosing, animals were sacrificed and metastatic lungtumor nodules were counted. Data is graphed as mean lung nodulecount±SEM.

As shown in FIG. 40, treatment of animals with anti-TGFβ antibodies 1D11and 28G11, but not 16B4, resulted in a statistically significantreduction of metastatic lung nodules relative to isotype controlantibody treated animals (p<0.05, unpaired T test following removal ofoutliers). These data demonstrate that the two anti-LAP antibodies 28G11and 16B4 have different functional effects in a mouse model of tumormetastasis. The finding that 28G11 has comparable efficacy to theanti-TGFβ antibody 1D11 is consistent with the effects of 28G11 beingdue to effects on the TGFβ pathway.

Example 28: Efficacy of Anti-LAP Antibodies in the CT26 Syngeneic Modelin Combination with Radiation

This Example describes the efficacy of anti-LAP antibodies incombination with radiation in a syngeneic CT26 tumor model.

Briefly, 1×10⁶ CT26 colorectal cancer cells were implanted into 6-8week-old Balb/c mice. Eight days after implantation, animals wererandomized into 6 groups of 16 animals each when mean tumor volume was300 mm² (day 0). Starting on day 0, animals were dosed with mouse IgG2aisotype control antibody (Group 1), anti-LAP antibody 28G11-IgG2a (Group2), 12 Gy radiotherapy and mouse IgG2a isotype control antibody (Group3), 20 Gy radiotherapy and mouse IgG2a isotype control antibody (Group4), 12 Gy radiotherapy and anti-LAP antibody 28G11-IgG2a (Group 5), or20 Gy radiotherapy and anti-LAP antibody 28G11-IgG2a (Group 6). Allantibodies were dosed intraperitoneally at 10 mg/kg. Groups 1 and 2received a total of 3 doses of antibody on days 0, 3, and 6 and thoseanimals were sacrificed at day 7 due to large tumor burden. Groups 3-6received a total of 5 doses of antibody on days 0, 3, 6, 9, and 12.Three random animals from Groups 3-6 were also sacrificed on day 7 andthe remaining animals were followed to day 19. In all cases whereanimals received radiation therapy, radiation was dosed only once on day0. Survival was assessed daily and tumor volumes were measured 3 timesper week by caliper using the formula V=W2×L/2. Data is presented asmean tumor volume+/−SEM of surviving animals.

As shown in FIGS. 41A and 41B, treatment of animals with 12 or 20 Gyradiation alone resulted in a delay in tumor growth. Co-administrationof 28G11 at 12 Gy radiation dose resulted in a statistically significantreduction in tumor growth relative to radiation treatment alone(****P<0001, ***P=0004, 2-way ANOVA). Co-administration of 28G11 at 20Gy radiation dose also resulted in a reduction relative to radiationtreatment alone, and that effect also was statistically significant.

Example 29: Effects of Anti-LAP Antibodies on CD73 Expression

In this Example, the effect of anti-LAP antibodies on CD73 expression inthe tumor microenvironment was examined. CD73 is a cell surface enzymethat processes adenosine monophosphate (AMP) to adenosine, a moleculewith known immunosuppressive effects in the tumor microenvironment.

CT26 tumors were grown in Balb/c mice to 300 mm² (designated day 0), andantibody 28G11 was dosed at 10 mg/kg on days 0, 3, and 6. Mice weretreated with targeted radiation (12 Gy or 20 Gy) at a single dose on day0. CD73 expression on monocytic myeloid-derived suppressor cells(mMDSCs), M2 macrophages, and dendritic cells was examined by flowcytometry on day 7 after radiation. Groupings were as follows:

Group 1: isotype control, no radiation (n=5)

Group 2: 28G11, no radiation (n=5)

Group 3: isotype control, 12 Gy radiation (N=3)

Group 4: isotype control, 20 Gy radiation (N=3)

Group 5: 28G11, 12 Gy radiation (N=2)

Group 6: 28G11, 20 Gy radiation (N=3)

As shown in FIGS. 42A-42C, radiation at both doses (12 Gy and 20 Gy)induced CD73 expression on mMDSCs, M2 macrophages, and dendritic cells.This increase in CD73 expression was attenuated by treatment with 28G11.Moreover, 28G11 reduced CD73 expression to below baseline levels inmMDSCs of mice which were not treated with radiation (FIG. 42A). Theseresults demonstrate that anti-LAP antibody treatment reduced both thenumber and immunosuppressive ability of inhibitory cell populations, asreflected in the reduced proportion of CD73 positive mMDSCs, M2macrophages, and dendritic cells.

Example 30: Biodistribution of Anti-LAP Antibodies

In this Example, the biodistribution of anti-LAP antibodies in miceharboring tumors was examined.

Briefly, 3 Balb/C mice were implanted with 1×10⁶ CT26 cells and tumorswere allowed to grow until they reached a mean tumor volume of 150 mm³.Animals were dosed with a single injection of 28G11_hIgG1 at 10 mg/kg.Three days post-injection, animals were sacrificed and blood wascollected. Mice were perfused with PBS and heart, liver, kidney, bone,colon, lung, and spleen tissue were harvested. Tissue was placed in 10%Neutral buffered formalin, stored overnight at 4° C., and transferred to80% ethanol. Tissue samples were sectioned and stained with anti-humanIgG1 to identify the location of 28G11 within the animal. There wasminimal staining observed in most tissues, with the strongest stainingobserved in tumor tissue.

Example 31. Efficacy of 20E6 and 28G11 Alone and in Combination withAnti-PD-1 in Animal Models of Cancer

This Example describes the testing the efficacy of 20E6 and 28G11antibodies alone and in combination with anti-PD-1 in the EMT6 mousebreast cancer tumor model. The antibodies used are listed below:

-   -   Mouse×[LAP-TGFb1_H] mAh (28G11_VH_N56Q) mIgG2a/Kappa (CX):        28G11_mIgG2a    -   Mouse×[LAP-TGFb 1_H] mAh (20E6_Q1E_N54Q) IgG2a/Kappa (CX):        20E6_mIgG2a    -   Mouse×[HEXON_Ad] mAh (TC31.27F11,C2) IgG2a/Kappa (CC): isotype        control antibody

Briefly, 6-8 weeks-old Balb/c mice were inoculated subcutaneously with0.3×10⁶ EMT6 mouse breast cancer cells. Animals were stratified into 6treatment groups of 10 animals each when the tumors grew to an averagesize of ˜85 mm³, at which point treatment was initiated. All antibodieswere administered intraperitoneally. Antibodies 20E6 and 28G11 weredosed at 10 mg/kg twice per week, while anti-PD 1 was dosed at 5 mg/kgevery 5 days. The vehicle-control group consisted of a murine IgG1isotype control dosed at 5 mg/kg, and a murine IgG2a isotype controldosed at 10 mg/kg. Tumors were measured 2-3 times per week and tumorvolume was calculated using the following formula: V=(tumorwidth)²×(tumor length)/2. It was observed that treatment of subjectswith antibody 20E6 and antibody 28G11 alone resulted in significanttumor growth inhibition compared to subjects treated with the isotypecontrol antibody. Furthermore, the combination treatment of eitherantibody 20E6 or antibody 28G11 with anti-PD-1 antibody resulted in 6complete responses where animals did not have any residual tumors. SeeFIGS. 43A-43H. All treatments were observed to be well tolerated and didnot cause any bodyweight loss.

Example 32: Binding of Anti-LAP F(Ab′) to Human LAP-TGFβ Isoforms 1, 2,and 3

To avoid the interference of avidity in the affinity measurement, thisExample analyzed the binding kinetics of 20E6 F(ab′) binding protein tohuman LAP-TGFβ. This Example describes the isoform specificity ofhumanized 20E6 F(ab′) binding protein to bind to human LAP-TGFβ isoforms1, 2, and 3 using surface plasmon resonance.

A Series S CM4 sensor chip (GE Healthcare, catalog BR100534) wasimmobilized with an anti-human Fc capture antibody following the kitprotocol (GE Healthcare, catalog BR100839) on a Biacore T200 instrumentwith 1×HBS-EP+ (Teknova, catalog H8022). Kinetic binding interactionsbetween human LAP-TGFβ isoforms 1, 2, and 3 and humanized 20E6 F(ab′)were performed in 1×HBS-EP+ with 0.1 mg/mL BSA (Jackson Immunoresearch,catalog 001-000-162) at 25° C. Approximately 50-65 RU of humanLAP-TGFβ-Fc isoforms were captured to the anti-human Fc surface followedby injection of 1:3 serially diluted humanized 20E6 F(ab′) from 3000 nMto 1.37 nM and including 0 nM F(ab′). The binding data was doublereferenced by subtraction of signal from a reference (capture surfaceonly) flow cell and the 0 nM F(ab′) injection. Binding rate constantswere determined by fitting the data with a 1:1 binding model (GEHealthcare Biacore T200 Evaluation software 2.0).

As shown in the figures and Table 32, the humanized 20E6 IgG1 antibody,when captured to the anti-human Fc capture sensor chip, exhibited anon-1:1 binding profile due to the bivalent nature of both the IgG1 andbivalent epitopes presented by the LAP-TGFβ1 molecule. See FIG. 44A.Also it was observed that monovalent humanized 20E6 F(ab′) bound withnanomolar affinity to human LAP-TGFβ1 (FIG. 44B), but no appreciablesignal increase was observed for human LAP-TGFβ2 (FIG. 44C) or LAP-TGFβ3(FIG. 44D). These data demonstrate that the humanized 20E6 F(ab′)specifically bound to human LAP-TGFβ1.

TABLE 32 Binding parameters for humanized 20E6_F(ab′) binding to humanTGFβ isoforms k_(on) k_(off) K_(D) Isoform (×10⁶ M⁻¹s⁻¹) (×10⁻² s⁻¹)(nM) LAP-TGFβ1 1.96 8.00 40.8 LAP-TGFβ2 — — — LAP-TGFβ3 — — —

Example 33: Binding of Anti-LAP F(Ab′) to Human, Cynomolgus Monkey, Rat,and Mouse LAP-TGFβ1

This Example describes the species specificity of humanized 20E6 F(ab′)binding protein to bind to LAP-TGFβ1 from several species using surfaceplasmon resonance.

A Series S CM4 sensor chip (GE Healthcare, catalog BR100534) wasimmobilized with an anti-human Fc capture antibody following the kitprotocol (GE Healthcare, catalog BR100839) on a Biacore T200 instrumentwith 1×HBS-EP+ (Teknova, catalog H8022). Kinetic binding interactionsbetween human, cynomolgus monkey, rat, and mouse LAP-TGFβ1and humanized20E6 F(ab′) were performed in 1×HBS-EP+ with 0.1 mg/mL BSA (JacksonImmunoresearch, catalog 001-000-162) at 25° C. Approximately 60-95 RU ofhuman, cynomolgus monkey, rat, and mouse LAP-TGFβ-Fc were captured tothe anti-human Fc surface followed by injection of 1:3 serially dilutedhumanized 20E6 F(ab′) from 3000 nM to 1.37 nM and including 0 nM F(ab′).The binding data was double referenced by subtraction of signal from areference (capture surface only) flow cell and the 0 nM F(ab′)injection. Binding rate constants were determined by fitting the datawith a 1:1 binding model (GE Healthcare Biacore T200 Evaluation software2.0).

As shown in the figures and Table 33, humanized 20E6 F(ab′) bound withnanomolar affinity to human LAP-TGFβ1 (FIG. 45A), cynomolgus monkeyLAP-TGFβ1 (FIG. 45B), ratLAP-TGFβ1 (FIG. 45C), and mouse LAP-TGFβ1 (FIG.45D).

TABLE 33 Binding parameters for humanized 20E6 F(ab′) binding toLAP-TGFβ1 from multiple species k_(on) k_(off) K_(D) Species (×10⁶M⁻¹s⁻¹) (×10⁻² s⁻¹) (nM) Human 1.91 ± 0.53 9.39 ± 2.35 49.5 ± 1.4Cynomolgus 1.58 ± 0.03 9.50 ± 0.12 60.3 ± 0.9 Rat 1.79 ± 0.42 10.6 ±2.57 59.4 ± 0.7 Mouse 1.64 ± 0.04 7.90 ± 0.11 48.3 ± 0.9 All valuesreported as average ± standard deviation from triplicate measurements

Example 34: Inhibition of Integrin (Avb6) Activation of LAP-TGFb1 with

This Examples examined the inhibition of integrin (avb6) activation ofLAP-TGFb1 using a LAP antibody 20E6_mIgG2a. Recombinant human αVβ6integrin (R&D Systems; cat. #3817-AV) was coated in a 96 well flatbottom tissue culture plate at 2 ug/ml in serum free RPMI for 2 hours at37C. Wells were treated with a 3-fold serial dilution (high of 30 ug/ml)of 20E6_mIgG2a, isotype control or anti-αVβ6 (10D5; commerciallyavailable from Millipore Sigma). Immediately after treatment P3U1 cellsexpressing human LAP-TGFβ1 (5×10⁴/well) were added to the plate followedby HEK-Blue TGFβ (2×10⁴/well) cells. (HEK-Blue TGFβ cells contain a SMADbinding element responsive SEAP reporter resulting in secretion of SEAPwhen bioactive TGFβ binds to receptor.) The plates were then incubatedovernight at 37° C. and the following day 125 μL of supernatant wastaken and plated in a 96-well v-bottom plate and spun again at 500G for5 minutes to remove cells. Secreted Alkaline Phosphatase (SEAP) levelsin the supernatant (25 μL) were detected utilizing the Great EscAPeChemiluminescence Kit 2.0 (Takara Bio; cat. #631736) according to themanufacturer protocol. Data show that the 20E6_mIgG2a effectivelyinhibited integrin avb6 activation of LAP-TGFb1 as compared to theisotype control antibody and the anti-αVβ6 (10D5) antibody (FIG. 46).

TABLE 34  Summary table of sequences SEQ ID Description SequenceProtein sequences   1 human LAP-TGFβ1LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS   2LAP region of human LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLAP-TGFβ1 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHR   3 human LAP-TGβ2LSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMI VKSCKCS   4LAP region of human LSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVLAP-TGFβ2 PPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRK KNSGKTPHLLLMLLPSYRLESQQTNRRKKR   5human LAP-TGFβ3 LSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCK CS   6 LAP region of humanLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVP LAP-TGFβ3YQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILM MIPPHRLDNPGQGGQRKKR   7mouse LAP-TGFβ1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS   8LAP region of mouse LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLAP-TGFβ1 LPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRR   9 mouse LAP-TGFβ2LSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPDEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPSHLPSENAIPPTFYRPYFRIVRFDVSTMEKNASNLVKAEFRVFRLQNPKARVAEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVQEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYASGDQKTIKSTRKKTSGKTPHLLLMLLPSYRLESQQSSRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHTKVLSLYNTINPEASASPCCVSQDLEPLTILYYIGNTPKIEQLSNMI VKSCKCS  10LAP region of mouse LSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPDEVLAP-TGFβ2 PPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPSHLPSENAIPPTFYRPYFRIVRFDVSTMEKNASNLVKAEFRVFRLQNPKARVAEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVQEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYASGDQKTIKSTRKKTSGKT PHLLLMLLPSYRLESQQSSRRKKR  11mouse LAP-TGFβ3 LSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPSVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQETSESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNGTNLFRAEFRVLRVPNPSSKRTEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENVHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDSPGQGSQRKKRALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCK CS  12 LAP region of mouseLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPSVMTHVP LAP-TGFβ3YQVLALYNSTRELLEEMHGEREEGCTQETSESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNGTNLFRAEFRVLRVPNPSSKRTEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNGDILENVHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILM MIPPHRLDSPGQGSQRKKR  13human LAP-TGFβ1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP“open conformation” LPEAVLALYNSTRDRVAGESAEPEPEPEADTYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS  14human LAP-TGFβ1 LSTCKTIDMELVKRKRIEAIRGQILSCLRLASPPSQGEVPPGP“closed conformation” LPEAVLALYNSTRDRVAGESAEPEPEPEADYCAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS  15human free TGFβ1 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANF(mature TGFβ1 without CLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPL LAP)PIVYYVGRKPKVEQLSNMIVRSCKCS 257 Human LAP-TGFβ1 inLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGP FIG. 34LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINAGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCSBinding protein, antibody and antigen binding fragments  1628G11(hyb)VHCDR1 DYYMS  17 28G11(hyb)VHCDR2 FIRNKPNGYTTE(extended definition of VHCDR2 used for humanization)  1828G11(hyb)VHCDR3 YTGGGYFDY  19 28G11(hyb)VLCDR1 RASQDISNYLN  2028G11(hyb)VLCDR2 YTSRLHS  21 28G11(hyb)VLCDR3 QQGDTLPWT  2228G11(hyb) VH EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLGFIRNKPNGYTTEYSASVKGRFTISRDNSQSILYLQMNVLRAEDSATYYCARYTGGGYFDYWGQGTTLTVSS  23 28G11(hyb) VLDIQMTQTTSSLSASLGDRLTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQADIATY FCQQGDTLPWTFGGGTKLEIK  2428G11(hyb) heavy chain EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLGFIRNKPNGYTTEYSASVKGRFTISRDNSQSILYLQMNVLRAEDSATYYCARYTGGGYFDYWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTD SFSCNVRHEGLKNYYLKKTISRSPGK 25 28G11(hyb) light chain DIQMTQTTSSLSASLGDRLTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQADIATYFCQQGDTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC  26 28G11 VHCDR2 N56QFIRNKPQGYTTE  27 28G11 VHCDR2 N56S FIRNKPSGYTTE  28 28G11 VHCDR2 N56HFIRNKPHGYTTE  29 28G11 VHCDR2 N56L FIRNKPLGYTTE  30 28G11 VHCDR2 N56DFIRNKPDGYTTE  31 28G11_H0 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKPNGYTTEYSASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSS  32 28G11_H0_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVGFIRNKPNGYTTEYSASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  3328G11_H1 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPNGYTTEYSASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSS  34 28G11_H1_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPNGYTTEYSASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  3528G11_H2 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPNGYTTEYSASVKGRFTISRDNSQSSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLLTVSS  36 28G11_H2_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPNGYTTEYSASVKGRFTISRDNSQSSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  3728G11_H2_IgG4mut EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPNGYTTEYSASVKGRFTISRDNSQSSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK  3828G11_H2.1 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPQGYTTEYSASVKGRFTISRDNSQSSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLLTVSS  39 28G11_H2.1_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWLGFIRNKPQGYTTEYSASVKGRFTISRDNSQSSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  4028G11_H2a VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIRNKPNGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSS  41 28G11_H2a_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIRNKPNGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  4228G11_H2b VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIRNKPQGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSS  43 28G11_H2b_IgG1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIRNKPQGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK  4428G1_H2b_hIgG4mut EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGK VHGLEWVGFIRNKPQGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSS  45 28G11_H2b_hIgG4mutEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAPGKGLEWVGFIRNKPQGYTTEYSASVKGRFTISRDNSKNSLYLQMNSLKTEDTAVYYCARYTGGGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK  4628G11_L1 VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATY FCQQGDTLPWTFGQGTKLEIK  4728G11_L1 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  48 28G11_L2 VLDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATY FCQQGDTLPWTFGQGTKLEIK  4928G11_L2 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  50 28G11_L3 VLDIQMTQSPSSLSASVGDRLTISCRASQDISNYLNWYQQKPGKTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATY FCQQGDTLPWTFGQGTKLEIK  5128G11_L3 DIQMTQSPSSLSASVGDRLTISCRASQDISNYLNWYQQKPGKTVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYFCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  52 28G11_L3a VLDIQMTQSPSSLSASVGDRLTISCRASQDISNYLNWYQQKPGKVVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATY YCQQGDTLPWTFGQGTKLEIK  5328G11_L3a DIQMTQSPSSLSASVGDRLTISCRASQDISNYLNWYQQKPGKVVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  54 22F9(hyb)VHCDR1GYTFTSYWMH  55 22F9(hyb)VHCDR2 MIHPNSGSTN  56 22F9(hyb)VHCDR3 YDYDGFFDV 57 22F9(hyb)VLCDR1 RASKSVSTSGYSYMH  58 22F9(hyb)VLCDR2 LASNLES  5922F9(hyb)VLCDR3 QHSRELPYT  60 22F9(hyb) VHQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAYYDYDGFFDVWGTGTTVTVSS  61 22F9(hyb) VLDIVLTQSPASLDVSLGQRATISCRASKSVSTSGYSYMHWYQQKSGQPPKLLIYLASNLESGVPARFSGSGSGTHFTLNIHPVEEED AATYYCQHSRELPYTFGGGTKLEIK 62 22F9(hyb) heavy chain QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAYYDYDGFFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNH HTEKSLSHSPGK  6322F9(hyb) light chain DIVLTQSPASLDVSLGQRATISCRASKSVSTSGYSYMHWYQQKSGQPPKLLIYLASNLESGVPARFSGSGSGTHFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NEC  64 22F9 VHCDR2 N54AMIHPASGSTN  65 22F9 VHCDR2 N54H MIHPHSGSTN  66 22F9 VHCDR2 N54QMIHPQSGSTN  67 22F9 VHCDR2 N54S MIHPSSGSTN  68 22F9 VHCDR3 D102AYDYAGFFDV  69 22F9 VHCDR3 D102E YDYEGFFDV  70 22F9 VHCDR3 D102GYDYGGFFDV  71 22F9_H0 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYDYDGFFDVWGQGTLVTVSS  72 22F9_H0_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  7322F9_H0.1 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWMGMIHPqSGSTNYNEKFKSkaTlTRDkSsSTaYMELSSLRSEDTAVYYCARYDYaGFFDVWGQGTLVTVSS  74 22F9_H0.1_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWMGMIHPqSGSTNYNEKFKSkaTlTRDkSsSTaYMELSSLRSEDTAVYYCARYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  7522F9_H1 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKEKSRVTMTvDTSTSTVYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSS  76 22F9_H1_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKEKSRVTMTvDTSTSTVYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  7722F9_H1.1 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKFKSkaTMTVDkSsSTVYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSS  78 22F9_H1.1_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKEKSkaTMTVDkSsSTVYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  7922F9_H1.1_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKFKSkaTMTVDkSsSTVYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK  8022F9_H2 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKFKSRVTlTvDTSTSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSS  81 22F9_H2_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKEKSRVTlTvDTSTSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  8222F9_H2.1 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKFKSkaTLTVDkSTSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSS  83 22F9_H2.1_IgG1QVQLVQpGAEVVKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKFKSkaTLTVDkSTSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  8422F9_H2.1_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWIGMIHPqSGSTNYNEKFKSkaTLTVDkSTSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVENAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK  8522F9_H3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKFKSRVTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSS  86 22F9_H3_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPNSGSTNYNEKFKSRVTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  8722F9_H3.1 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPqSGSTNYNEKFKSRVTLTVDKSSSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSS  88 22F9_H3.1_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPqSGSTNYNEKFKSRVTLTVDKSSSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  8922F9_H3.1_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPqSGSTNYNEKFKSRVTLTVDKSSSTAYMELSSLRSEDTAVYYCAYYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVENAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK  9022F9_H4 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPNSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSS  91 22F9_H4_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPNSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  9222F9_H5 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPNSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSS  93 22F9_H5_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPNSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  9422F9_H5_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPNSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYDGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK  9522F9_H5.2 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPQSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYAGFFDVWGQGTLVTVSS  96 22F9_H5.2_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPQSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYAGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK  9722F9_H5.2_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGMIHPQSGSTNYNEKFKSkaTlTvDkSsSTaYMELSSLRSEDTAVYYCAyYDYAGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK  9822F9_H7 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSS  99 22F9_H7_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 10022F9_H7_IgG4mut QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQENWYVDGVEVENAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 10122F9_H7a VH EVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSS 102 22F9_H7a_IgG1EVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 10322F9_H7a_IgG4mut EVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVRQAPGQGLEWiGMIHPqSGSTNYAQKFQGRVTMTvDkSTSTVYMELSSLRSEDTAVYYCAyYDYaGFFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 10422F9_L0 VL DIQLTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQHSRELPYTFGGGTKVEIK105 22F9_L0 DIQLTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 106 22F9_L1 VLDIQLTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKpPKLLIYLASNLESGVPSRFSGSGSGThFTLTISSLQPED FATYYCQHSRELPYTFGGGTKVEIK107 22F9_L1 DIQLTQSPSSLSASVGDRVTITCRASKSVSTSGYSYMHWYQQKPGKpPKLLIYLASNLESGVPSRFSGSGSGThFTLTISSLQPEDFATYYCQHSRELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 108 22F9_L2 VLDIQLTQSPSSLSvSVGDRaTITCRASKSVSTSGYSYMHWYQQKPGKpPKLLIYLASNLESGVPSRFSGSGSGThFTLTISSvQPED FATYYCQHSRELPYTFGGGTKlEIK109 22F9_L2 DIQLTQSPSSLSvSVGDRaTITCRASKSVSTSGYSYMHWYQQKPGKpPKLLIYLASNLESGVPSRFSGSGSGThFTLTISSvQPEDFATYYCQHSRELPYTFGGGTKlEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 110 20E6(hyb)VHCDR1GYTFTSYWMH 111 20E6(hyb)VHCDR2 RIDPNSGGIK 112 20E6(hyb)VHCDR3 WDYGGYFDV113 20E6(hyb)VLCDR1 RASQDITNYLN 114 20E6(hyb)VLCDR2 YTSRLHS 11520E6(hyb)VLCDR3 QQGDTLPWT 116 20E6(hyb) VHQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGIKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWDYGGYFDVWGTGTTVTVSS 117 20E6(hyb) VLDIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGAVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATY FCQQGDTLPWTFGGGTKLEIK 11820E6(hyb) heavy chain QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGIKYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARWDYGGYFDVWGTGTTVTVSSSQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYN VSLIMSDTGGTCY 11920E6(hyb) light chain DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGAVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 120 20E6(hyb)VHCDR2 N54QRIDPQSGGIK 121 20E6(hyb)VHCDR2 N54G RIDPGSGGIK 122 20E6(hyb)VHCDR2 N54ARIDPASGGIK 123 20E6(hyb)VHCDR2 N54S RIDPSSGGIK 124 20E6(hyb)VHCDR2 N54HRIDPHSGGIK 125 20E6(hyb)VHCDR2 N54L RIDPLSGGIK 126 20E6(hyb)VHCDR2 N54DRIDPDSGGIK 127 20E6_H0 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYNEKFKSRVTMTRDTSISTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 128 20E6_H0_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYNEKFKSRVTMTRDTSISTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 12920E6_H0_IgG4mut QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYNEKFKSRVTMTRDTSISTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 13020E6_H0.1 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 131 20E6_H0.1_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 13220E6_H0.1_IgG4mut QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPNSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 13320E6_H0.2 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 134 20E6_H0.2_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 13520E6_H0.2_IgG4mut QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 13620E6_H0.3_IgG1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPgSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 137 20E6_H0.4_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPaSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 138 20E6_H0.5_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPsSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 139 20E6_H0.6_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPhSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 140 20E6_H0.7_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPlSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 141 20E6_H0.8_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPdSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 142 20E6_H0.2_hIgG4mutQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQ VHGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 143 20E6_H0.2_hIgG4mutQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 14420E6_H1 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGRIDPNSGGIKYNEKFKSRVTlTvDTSISTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 145 20E6_H1_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGRIDPNSGGIKYNEKFKSRVTlTvDTSISTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 14620E6_H2 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGRIDPNSGGIKYNEKFKSRVTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 147 20E6_H2_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWiGRIDPNSGGIKYNEKFKSRVTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 14820E6_H3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVkQrPGQGLEWiGRIDPNSGGIKYNEKEKSkaTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 149 20E6_H3_IgG1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVkQrPGQGLEWiGRIDPNSGGIKYNEKFKSkaTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 15020E6_H4 VH QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGRIDPNSGGIKYNEKFKSkaTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 151 20E6_H4_IgG1QVQLVQpGAEVvKPGASVKlSCKASGYTFTSYWMHWVkQrPGQGLEWiGRIDPNSGGIKYNEKFKSkaTlTvDkSsSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 15220E6_L0 VL DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQGDTLPWTFGQGTKLEIK 15320E6_L0 DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 154 20E6_L1 VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAvKLLIYYTSRLHSGVPSRFSGSGSGTDyTLTISSLQPEDFATY fCQQGDTLPWTFGQGTKLEIK 15520E6_L1 DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAvKLLIYYTSRLHSGVPSRFSGSGSGTDyTLTISSLQPEDFATYfCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 156 20E6_L0_P44V VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQGDTLPWTFGQGTKLEIK 15720E6_L0 P44V DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 158 20E6_L0_F71Y VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATY YCQQGDTLPWTFGQGTKLEIK 15920E6_L0_F71Y DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 160 20E6_L0_Y87F VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY FCQQGDTLPWTFGQGTKLEIK 16120E6_L0_Y87F DIQMTQSPSSLSASVGDRVTITCRASQDITNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 17G8(hyb)VHCDR1GFTFTDYYMS 163 17G8(hyb)VHCDR2 FIRNKANGYTTE 164 17G8(hyb)VHCDR3YKLGGYFDV 165 17G8(hyb)VLCDR1 RASQDISNYLN 166 17G8(hyb)VLCDR2 YTSRLHS167 17G8(hyb)VLCDR3 QQGNTLPWT 168 17G8(hyb) VHEVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNALRAEDSATYYCARYKLGGYFDVWGTGTTVTVSS 169 17G8(hyb) VLDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATY FCQQGNTLPWTFGGGTKLEIK 17017G8(hyb) heavy chain EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNALRAEDSATYYCARYKLGGYFDVWGTGTTVTVSSSQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTL YNVSLIMSDTGGTCY 17117G8(hyb) light chain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 172 17G8(hyb)VHCDR2 N54QFIRNKAQGYTTE 173 17G8(hyb)VHCDR2 N54G FIRNKAGGYTTE 17417G8(hyb)VHCDR2 N54A FIRNKAAGYTTE 175 17G8(hyb)VHCDR2 N54S FIRNKASGYTTE176 17G8(hyb)VHCDR2 N54H FIRNKAHGYTTE 177 17G8(hyb)VHCDR2 N54LFIRNKALGYTTE 178 17G8(hyb)VHCDR2 N54D FIRNKADGYTTE 179 24E3(hyb)VHCDR1GYTFTSYNMH 180 24E3(hyb)VHCDR2 AIYPGNGDTS 181 24E3(hyb)VHCDR3 WDWGGYFDV182 24E3(hyb)VLCDR1 RASQDISNYLN 183 24E3(hyb)VLCDR2 YTSRLHS 18424E3(hyb)VLCDR3 QQGDTLPWTF 185 24E3(hyb)VHQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNPKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRWDWGGYFDVWGTGTTVTVSS 186 24E3(hyb) VLDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATY FCQQGDTLPWTFGGGTKLGIK 18724E3(hyb) heavy chain QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNPKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCTRWDWGGYFDVWGTGTTVTVSSSQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYN VSLIMSDTGGTCY 18824E3(hyb) light chain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTLPWTFGGGTKLGIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 189 24E3(hyb)VHCDR2 N54QAIYPGQGDTS 190 24E3(hyb)VHCDR2 N54G AIYPGGGDTS 191 24E3(hyb)VHCDR2 N54AAIYPGAGDTS 192 24E3(hyb)VHCDR2 N54S AIYPGSGDTS 193 24E3(hyb)VHCDR2 N54HAIYPGHGDTS 194 24E3(hyb)VHCDR2 N54L AIYPGLGDTS 195 24E3(hyb)VHCDR2 N54DAIYPGDGDTS 196 Human IgG1 constantASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS regionGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 197 Human IgG4 variantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS constant regionGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD (also referred to as HKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKD “IgG4mut”)TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK198 chicken LAP-TGFβ1 LSTCQRLDLEAAKKKRIEAVRGQILSKLRLTAPPPASETPPRPLPDDVRALYNSTQELLKQRARLRPPPDGPDEYWAKELRRIPMETTWDGPMEHWQPQSHSIFFVFNVSRVRAEVGGRALLHRAELRMLRQKAAADSAGTEQRLELYQGYGNASWRYLHGRSVRATADDEWLSFDVTDAVHQWLSGSELLGVFKLSVHCPCEMGPGHADEMRISIEGFEQQRGDMQSIAKKHRRVPYVLAMALPAERANELHSARRRRDLDTDYCFGPGTDEKNCCVRPLYIDFRKDLQWKWIHEPKGYMANFCMGPCPYIWSADTQYTKVLALYNQHNPGASAAPCCVPQTLDPLPIIYYVGRNVRVEQLSNMVVRACKCS 199 human-chicken LAP-LSTCQRLDLEAAKKKRIEAVRGQILSKLRLTAPPPASETPPRP TGFβ1 chimera #1LPDDVRALYNSTQELLKQRARLRPPPDGPDEYWAKELRRIPMETTWDEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCS 200human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLTAPPPASETPPRPTGFβ1 chimera #1.2 LPDDVRALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS 201human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #1.3 LPEAVLALYNSTQELLKQRARLRPPPDGPDEYWAKELRRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCS 202human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #2 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNGPMEHWQPQSHSIFFVFNVSRVRAEVGGRALLHRAELRMLRQKAAADSAGTEQRLELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVY YVGRKPKVEQLSNMIVRSCKCS 203human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #2.1 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRIPMETTWDGPMEHWQPQSHSIFFVFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRK PKVEQLSNMIVRSCKCS 204human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #2.2 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNVSRVRAEVGGRALLHRAELRMLRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYV GRKPKVEQLSNMIVRSCKCS 205human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #2.3 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRMLRQKAAADSAGTEQRLELYQGYGNASWRYLHGRSVRATADDEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVY YVGRKPKVEQLSNMIVRSCKCS 206human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #3 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQGYGNASWRYLHGRSVRATADDEWLSFDVTDAVHQWLSGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKP KVEQLSNMIVRSCKCS 207human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #4 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGSELLGVFKLSVHCPCEMGPGHADEMRISIEGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYY VGRKPKVEQLSNMIVRSCKCS 208human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #5 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFEQQRGDMQSIAKKHRRVPYVLAMALPAERANELHSARRRRDLDTDYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCS 209human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #6 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFGPGTDEKNCCVRPLYIDFRKDLQWKWIHEPKGYMANFCMGPCPYIWSADTQYTKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVG RKPKVEQLSNMIVRSCKCS 210human-chicken LAP- LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera #7 LPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQTLDPLPIIYYVGRN VRVEQLSNMVVRACKCS 211Chicken-human LAP- LSTSQRLDLEAAKKKRIEAVRGQILSKLRLTAPPPASETPPRPTGFβ1 chimera LPDDVRALYNSTQELLKQRARLRPPPDGPDEYWAKELRRVLMV chB1ex2.1_2.2ETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYGNASWRYLHGRSVRATADDEWLSFDVTDAVHQWLSGSELLGVFKLSVHCPCEMGPGHADEMRISIEGFEQQRGDMQSIAKKHRRVPYVLAMALPAERANELHSARRRRDLDTDYCFGPGTDEKNCCVRPLYIDFRKDLQWKWIHEPKGYMANFCMGPCPYIWSADTQYTKVLALYNQHNPGASAAPCCVPQTLDPLPI IYYVGRNVRVEQLSNMVVRACKCS 212Chicken-human LAP- LSTSQRLDLEAAKKKRIEAVRGQILSKLRLTAPPPASETPPRPTGFβ1 chimera LPDDVRALYNSTRDRVAGESAEPEPEPEADYYAKEVTRIPMET chB1ex1.3TWDGPMEHWQPQSHSIFFVFNVSRVRAEVGGRALLHRAELRMLRQKAAADSAGTEQRLELYQGYGNASWRYLHGRSVRATADDEWLSFDVTDAVHQWLSGSELLGVFKLSVHCPCEMGPGHADEMRISIEGFEQQRGDMQSIAKKHRRVPYVLAMALPAERANELHSARRRRDLDTDYCFGPGTDEKNCCVRPLYIDFRKDLQWKWIHEPKGYMANFCMGPCPYIWSADTQYTKVLALYNQHNPGASAAPCCVPQTLD PLPIIYYVGRNVRVEQLSNMVVRACKCS213 Chicken-human LAP- LSTSQRLDLEAAKKKRIEAVRGQILSKLRLASPPSQGEVPPGPTGFβ1 chimera LPEAVLALYNSTQELLKQRARLRPPPDGPDEYWAKELRRIPME chB1_ex1.2TTWDGPMEHWQPQSHSIFFVFNVSRVRAEVGGRALLHRAELRMLRQKAAADSAGTEQRLELYQGYGNASWRYLHGRSVRATADDEWLSFDVTDAVHQWLSGSELLGVFKLSVHCPCEMGPGHADEMRISIEGFEQQRGDMQSIAKKHRRVPYVLAMALPAERANELHSARRRRDLDTDYCFGPGTDEKNCCVRPLYIDFRKDLQWKWIHEPKGYMANFCMGPCPYIWSADTQYTKVLALYNQHNPGASAAPCCVPQTLDPLPIIYYVGRNVRVEQLSNMVVRACKCS 214 Linker PVGVV 215 LAP-TGFβ1 epitopeVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAE 216 28G11_L0 VLDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQQGDTLPWTFGQGTKLEIK 21728G11_L0 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKVPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGDTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 218 20E6_H0.2a VHEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSS 219 20E6_H0.2a_IgG1EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 22020E6_H0.2a_IgG4mut EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIDPQSGGIKYAQKFQGRATLTVDTSTSTAYMELSRLRSDDTAVYYCARWDYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 2217H4_HC(hyb) DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSF SCNVRHEGLKNYYLKKTISRSPGK 2227H4_LC(hyb) DIQMTQSPSSLSASLGGKVTITCKASQDIDKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFNISNLEPEDIATYYCLQYDNLRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 223 7H4_VHDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSS 224 7H4_VLDIQMTQSPSSLSASLGGKVTITCKASQDIDKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFNISNLEPEDIATY YCLQYDNLRTFGGGTKLEIK 2257H4_HCDR1 GYSITSGYYWN 226 7H4_HCDR2 YISYDGTNNYNPSLKN 227 7H4_HCDR3SFYNNYFDF 228 7H4_LCDR1 KASQDIDKYIA 229 7H4_LCDR2 YTSTLQP 230 7H4_LCDR3LQYDNLRT 231 7H4_HCDR2 (D55G) YISYGGTNNYNPSLKN 232 7H4_HCDR2 (D55A)YISYAGTNNYNPSLKN 233 7H4_HCDR2 (D55E) YISYEGTNNYNPSLKN 2347H4_VHmut#1 (D55G) DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSS 235 7H4_VHmut#2 (D55A)DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYAGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSS 236 7H4_VHmut#3 (D55E)DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYEGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSS 237 7H4_HCmut#1 (D55G)DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSF SCNVRHEGLKNYYLKKTISRSPGK 2387H4_HCmut#2 (D55A) DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYAGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSF SCNVRHEGLKNYYLKKTISRSPGK 2397H4_HCmut#3 (D55E) DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYEGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSF SCNVRHEGLKNYYLKKTISRSPGK 240Pembrolizumab heavy QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQ chainGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 241Pembrolizumab light EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQK chainPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 242 20E6 VHCDR3WX₁YGGYFX₂X₃ consensus (X₁-X₃ can be any amino acid) 243 20E6 VLCDR3QQGDX₁LPWT consensus (X₁ can be any amino acid) 244Human_IgGl_P01857_L234A_L235AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 245 Human_IgG1_P01857_L234A_L235A_D265SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVsVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 246 Nivolumab heavy chainQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK 247Nivolumab light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 248 22F9_N54Q_D102AQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQ heavy chainGLEWIGMIHPQSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAYYDYAGFFDVWGTGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVH EGLHNHHTTKSFSRTPGK 24922F9_N54Q_D102A DIVLTQSPASLDVSLGQRATISCRASKSVSTSGYSYMHWYQQK light chainSGQPPKLLIYLASNLESGVPARFSGSGSGTHFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NEC 250Region 1 of LAP-TGFβ1 RKRIEAIRGQIL 251 Region 2 of LAP-TGFβ1 LASPPSQGEV252 Region 3 of LAP-TGFβ1 GWKWIHEPK 253 Region 4 of LAP-TGFβ1 YVGRKPK254 2F8 binding region VDINGFTTGRRGDLATIHGMN 255 7H4_HCmut #3 (D55G)DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGTNNYNPSLKNRISITRDTSKHQFFLKLNSVTTEDTATYYCARSFYNNYFDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSF SCNVRHEGLKNYYLKKTISRSPGK 256Kappa light chain VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNconstant domain ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In this Table and previous Tables, unless indicated otherwise, it isunderstood that underlined underlining indicates the CDRs in the bindingprotein (e.g., antibody or antigen binding fragment thereof). Note thata CDR might be defined and identified by any of the methods and systemsdescribed herein (e.g., Chothia, Kabat, and IMGT).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments disclosed herein. Such equivalents are intended to beencompassed by the following claims.

1. An isolated antibody or antigen binding fragment thereof whichspecifically binds to LAP comprising: (a) a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 110, 120, and 112, respectively, and a lightchain variable region comprising CDR1, CDR2, and CDR3 regions comprisingthe amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;(b) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and112, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 113, 114, and 115, respectively; (c) a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 16, 26, and 18, respectively, and a light chainvariable region comprising CDR1, CDR2, and CDR3 regions comprising theamino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; (d) aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57,58, and 59, respectively; (e) a heavy chain variable region comprisingCDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQID NOs: 54, 66, and 56, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 57, 58, and 59, respectively; (f) a heavy chainvariable region comprising CDR1, CDR2, and CDR3 regions comprising theamino acid sequences of SEQ ID NOs: 54, 55, and 68, respectively, and alight chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59,respectively; or (g) a heavy chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 54, 66, and 68, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 57, 58, and 59, respectively. 2-42. (canceled)43. A method of treating cancer comprising administering to a subject inneed thereof a therapeutically effective amount of an antibody orantigen binding fragment thereof, a bispecific molecule comprising theantibody or antigen binding fragment thereof or an immunoconjugatecomprising the antibody or antigen binding fragment thereof, wherein theantibody or antigen binding fragment thereof specifically binds to LAPand comprises: (a) a heavy chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110,120, and 112, respectively, and a light chain variable region comprisingCDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQID NOs: 113, 114, and 115, respectively; (b) a heavy chain variableregion comprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 110, 111, and 112, respectively, and a lightchain variable region comprising CDR1, CDR2, and CDR3 regions comprisingthe amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;(c) a heavy chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 16, 26, and18, respectively, and a light chain variable region comprising CDR1,CDR2, and CDR3 regions comprising the amino acid sequences of SEQ IDNOs: 19, 20, and 21, respectively; (d) a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chainvariable region comprising CDR1, CDR2, and CDR3 regions comprising theamino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively; (e) aheavy chain variable region comprising CDR1, CDR2, and CDR3 regionscomprising the amino acid sequences of SEQ ID NOs: 54, 66, and 56,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57,58, and 59, respectively: (f) a heavy chain variable region comprisingCDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQID NOs: 54, 55, and 68, respectively, and a light chain variable regioncomprising CDR1, CDR2, and CDR3 regions comprising the amino acidsequences of SEQ ID NOs: 57, 58, and 59, respectively; or (g) a heavychain variable region comprising CDR1, CDR2, and CDR3 regions comprisingthe amino acid sequences of SEQ ID NOs: 54, 66, and 68, respectively,and a light chain variable region comprising CDR1, CDR2, and CDR3regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and59, respectively.
 44. The method of claim 43, wherein the cancer ischaracterized by abnormal TGFβ activity.
 45. The method of claim 43,wherein the cancer is associated with infiltration of CD4+ regulatory Tcells, CD8+ regulatory T cells, regulatory B cells, myeloid-derivedsuppressor cells, tumor-associated macrophages, cancer-associatedfibroblasts, and/or innate lymphoid cells.
 46. The method of claim 43,wherein the cancer is selected from the group consisting of: breastcancer, bladder cancer, uterine/cervical cancer, ovarian cancer,prostate cancer, testicular cancer, esophageal cancer, gastrointestinalcancer, pancreatic cancer, colorectal cancer, colon cancer, kidneycancer, head and neck cancer, lung cancer, stomach cancer, germ cellcancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasmof the central nervous system, lymphoma, leukemia, myeloma, sarcoma, andmyelodysplastic syndromes.
 47. The method of claim 43, furthercomprising administering one or more additional therapies.
 48. Themethod of claim 47, wherein the one or more additional therapies isselected from radiation therapy, chemotherapy, an immune checkpointinhibitor, immunosuppressive therapy, immunostimulatory therapy, celltherapy, and a therapeutic agent.
 49. The method of claim 48, whereinthe immune checkpoint inhibitor is an anti-PD1 antibody, an anti-PD-L1antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGITantibody, or an anti-TIM3 antibody. 50-54. (canceled)
 55. The method ofclaim 43, wherein the antibody or antigen binding fragment thereofcomprises heavy and light chain variable region sequences which are atleast 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequencesselected from the group consisting of: (a) SEQ ID NOs: 218 and 154,respectively, (b) SEQ ID NOs: 133 and 154, respectively, (c) SEQ ID NOs:42 and 52, respectively; (d) SEQ ID NOs: 101 and 104, respectively; and(e) SEQ ID NOs: 98 and 104, respectively.
 56. The method of claim 43,wherein the antibody or antigen binding fragment thereof comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 218 or a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:218 with 1-5, 5-10, 10-15, 15-20, or 20-25 aminoacid substitutions; and a light chain variable region comprising theamino acid sequence of SEQ ID NO: 154 or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 154 with 1-5, 5-10,10-15, 15-20, or 20-25 amino acid substitutions.
 57. The method of claim43, wherein the antibody or antigen binding fragment thereof comprisesheavy and light chain variable region sequences selected from the groupconsisting of: (a) SEQ ID NOs: 218 and 154, respectively, (b) SEQ IDNOs: 133 and 154, (c) SEQ ID NOs: 42 and 52, respectively; (d) SEQ IDNOs: 101 and 104, respectively; and (e) SEQ ID NOs: 98 and 104,respectively.
 58. The method of claim 43, wherein the antibody orantigen binding fragment thereof comprises heavy and light chainsequences which are at least 85%, 90%, 95%, 98%, or 99% identical to theamino acid sequences selected from the group consisting of: (a) SEQ IDNOs: 219 and 155, respectively, (b) SEQ ID NOs: 220 and 155,respectively, (c) SEQ ID NOs: 134 and 155, respectively; (d) SEQ ID NOs:135 and 155, respectively, (e) SEQ ID NOs: 43 and 53, respectively; (f)SEQ ID NOs: 45 and 53, respectively; (g) SEQ ID NOs: 102 and 105,respectively; (h) SEQ ID NOs: 103 and 105, respectively; (i) SEQ ID NOs:99 and 105, respectively; and (j) SEQ ID NOs: 100 and 105, respectively.59. The method of claim 43, wherein the antibody or antigen bindingfragment thereof of claim 1 which comprises heavy and light chainsequences selected from the group consisting of: (a) SEQ ID NOs: 219 and155, respectively, (b) SEQ ID NOs: 220 and 155, respectively, (c) SEQ IDNOs: 134 and 155, respectively; (d) SEQ ID NOs: 135 and 155,respectively, (e) SEQ ID NOs: 43 and 53, respectively; (f) SEQ ID NOs:45 and 53, respectively; (g) SEQ ID NOs: 102 and 105, respectively; (h)SEQ ID NOs: 103 and 105, respectively; (i) SEQ ID NOs: 99 and 105,respectively; and (j) SEQ ID NOs: 100 and 105, respectively.
 60. Themethod of claim 43, wherein the antibody or antigen binding fragmentthereof comprises a heavy chain comprising amino acid sequences as setforth in SEQ ID NO: 218 and SEQ ID NO: 245 and a light chain comprisingan amino acid sequence as set forth in SEQ ID NO:
 155. 61. The method ofclaim 43, wherein the antibody or antigen binding fragment thereof bindsto human LAP, cyno LAP, rat LAP, and/or murine LAP.
 62. The method ofclaim 43, wherein the antibody or antigen binding fragment thereofinhibits TGFβ1 activation.
 63. The method of claim 43, wherein theantibody or antigen binding fragment thereof binds to human LAP with aK_(D) of 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20nM or less, or 10 nM or less.
 64. The method of claim 43, wherein theantibody or antigen binding fragment thereof binds to human LAP in theabsence of an anchor protein.
 65. The method of claim 43, wherein theantibody or antigen binding fragment thereof binds to immunosuppressivecells.
 66. The method of claim 43, wherein the antibody or antigenbinding fragment thereof binds to LAP complexed with an anchor proteinon immunosuppressive cells, but does not bind to the anchor protein orto an epitope composed of residues of both LAP and the anchor protein.67. The method of claim 66, wherein the anchor protein is GARP orLRRC33.
 68. The method of claim 65, wherein the immunosuppressive cellsare regulatory T cells, M2 macrophages, cancer cells expressing LAP,and/or myeloid-derived suppressor cells.
 69. The method of claim 43,wherein the antibody or antigen binding fragment thereof binds to bothGARP-positive immunosuppressive cells and GARP-negativeimmunosuppressive cells.
 70. The method of claim 43, wherein theantibody or antigen binding fragment thereof does not bind to LAP onextracellular matrix.
 71. The method of claim 43, wherein the antibodyor antigen binding fragment thereof does not bind to LAP complexed withLTBP1, LTBP3 and/or LTBP4.
 72. The method of claim 43, wherein theantibody or antigen binding fragment thereof comprises an IgG constantregion or variant thereof.
 73. The method of claim 43, wherein theantibody or antigen binding fragment thereof is a chimeric, human orhumanized antibody.
 74. The method of claim 43, wherein the antibody orantigen binding fragment thereof does not bind to human LAP comprising aY74T mutation or binds to human LAP comprising K27C and Y75C mutations.75. The method of claim 43, wherein the antibody or antigen bindingfragment thereof binds to one or more residues of residues 31-40,274-280, and 340-343 of human LAP-TGFβ1 (SEQ ID NO: 1), or binds to oneor more residues of residues 31-43, 272-283, and 340-344 of humanLAP-TGFβ1 (SEQ ID NO: 1).
 76. The method of claim 43, wherein thebispecific molecule is linked to a molecule having a second bindingregion and the second binding region binds to a tumor-associatedantigen.
 77. The method of claim 77, wherein the second binding regionbinds to CD4, CD8, CD45, CD56, CD14, CD16, CD19, CD11b, CD25, CD20,CD22, CD30, CD38, CD 114, CD23, CD73, CD163, CD206, CD203, CD200R, orCD39.
 78. The method of claim 43, wherein the immunoconjugate is linkedto a detectable moiety, a binding moiety, a labeling moiety, or abiologically active moiety.